TW201625680A - Humanized anti-Tau(pS422) antibody brain shuttles and use thereof - Google Patents

Abstract

Herein is reported a non-covalent complex of a haptenylated antibody that specifically binds to human Tau(pS422) and an anti-blood brain barrier receptor/hapten bispecific antibody.

Description

Humanized anti-Tau (pS422) antibody brain shuttle and its use

The present invention relates to a humanized anti-Tau (pS422) antibody brain shuttle construct that specifically binds to a phosphorylated Tau fragment of SEQ ID NO: 03 and which is useful for treating brain diseases.

Human Tau (microtubule-associated protein Tau (neuronal fiber entanglement protein, paired helical filament-Tau, PHF-Tau)) is a neuron microtubule-associated protein mainly present in axons and is used to promote tubulin Polymerize and stabilize microtubules. There are eight isoforms in the human brain (same isoforms A, B, C, D, E, F, G, fetus-Tau), and the longest isoform contains 441 amino acids (same isoforms) F, Uniprot P10636-8). Tau and its properties are also described by Reynolds, C.H., et al, J. Neurochem. 69 (1997) 191-198.

The phosphorylated form of Tau is a building block of neurofibrillary lesions in the brain of Alzheimer's disease (AD), a major component of paired helical filaments (PHF). Tau can be phosphorylated at its serine or threonine residues by several different kinases, including GSK3[beta], cdk5, MARK, and MAP kinase family members.

Tau proteinosis can be characterized by aberrant hyperphosphorylation of Tau and is based on Iqbal, K., et al. (Biochim. Biophys. Acta 1739 (2005) 198-210):

‧ Alzheimer's disease, including only the tangled form of the disease

‧Down syndrome, adult case

‧ Guam Parkinsonism dementia complex

‧ Boxing player dementia

‧Pick disease

‧ Silver granulosus dementia

‧ frontal lobe dementia

‧Cortical basal degeneration

‧ globus pallidus - pons - substantia nigra degeneration

‧Progressive nuclear paralysis

‧ entangled Gerstmann-Sträussler-Scheinker disease

To date, almost 40 serine (S)/threonine (T) phosphorylation sites have been found in Tau from the brain of Alzheimer's disease (Hanger, DP, et al., J. Biol. Chem .282 (2007) 23645-23654). The development of Tau lesions in Alzheimer's disease is related to its phosphorylation status. However, most of the 40 phosphorylation sites are not associated with disease lesions as they are also present in Tau extracted from healthy fetal brain tissue. Only a small amount of phosphorylation is unique to the disease condition and is probably responsible for defining the abnormal agglutination and specific insolubility of Tau in the PHF of Alzheimer's disease brain (Morishima-Kawashima, M., et al., J. Biol). Chem. 270 (1995) 823-829). According to Pei, J. J., et al. (J. Alzheimer's Disease 14 (2008) 385-392), information provided by existing literature on such sites specific for AD brain is limited and unclear. Pei used a list of Tau phosphorylation-specific antibodies and measured its content in the medial temporal cortex homogenate from 22 AD patients and 10 control groups.

Bussiere, T., et al. (Acta Neuropathol. 97 (1999) 221-230) describe phosphorylated serine 422 on Tau protein as found in several diseases associated with neuronal fiber degradation. Pathological epitopes. Augustinack, J.C., et al. (Acta Neuropathol. 103 (2002) 26-35) describe pS422 associated with the severity of neuronal disease in Alzheimer's disease. Guillozet-Bongaarts, A., (J. Neurochem. 97 (2006) 1005-1014) describes Tau phosphorylation at serine 422 as part of the maturation process of PHF. Tau pS422 was also found to be associated with the development of lesions in various transgenic mouse models of Alzheimer's disease. Thus, Deters, N., et al., Biochem. Biophys. Res. Commun. 379 (2009) 400-405, mentions that the double transgenic gene Dom5/pR5 mouse displays specifically phosphorylates the pathogenic S422 epitope. The number of hippocampal neurons in Tau is increased by a factor of seven. Goetz, J., et al. (Science 293 (2001) 1491-1495) report the appearance of Tau phosphorylated at S422 in the brain of Tau P301L transgenic mice injected with Abeta42 fibrils.

EP 2 009 104 relates to an epitope of a Tau protein present in a phosphorylated state in a Tau protein from Alzheimer's disease PHF and the use of the epitope, which is used to specifically detect Alzheimer's An antibody to the Tau protein. WO 2002/062851 and US 7,446,180 are related to antibodies which are specific for the abnormally truncated form of Tau protein and for the diagnostic and therapeutic aspects of Alzheimer's disease and related Tau protein disease.

WO 98/22120 is a method for treating a patient suffering from Alzheimer's disease, comprising administering to a patient for from about 207 to about 222 amino acids, from about 224 to about 240 amino acids and The step of phosphorylating the Tau fragment of about 390 to about 408 amino acids. Animal studies using vaccinated Tau fragment 379-408 [P-Ser396, 404] to vaccinate Tau transgenic mice are mentioned in Asuni, A. A., et al, J. Neuroscience 27 (2007) 9115-9129. US 2008/0050383 relates to a method of treating and preventing Alzheimer's disease or other Tau protein disease in an individual by administering a Tau protein fragment.

Hasegawa, M., et al. (FEBS Lett. 384 (1996) 25-30) reported a monoclonal antibody (AP422) specific for phosphoserine 422 in the microtubule-associated protein tau.

In WO 01/55725, antibodies that specifically recognize tau and antibodies that specifically recognize phosphorylated tau (181) are reported for in vivo diagnosis of tauopathy and/or in vivo differential diagnosis. Tau protein disease and non-tau protein disease methods.

In WO 02/027017, antibodies prepared from immunogens of polypeptides having phosphorylated serine are reported. WO 02/062851 is directed to antibodies specific for the abnormally truncated form of the Tau protein and for the diagnostic and therapeutic aspects of Alzheimer's disease and related Tau protein disease.

In WO 2004/016655, an antibody specific for the central nervous system (CNS) Tau protein is reported, wherein the antibody specifically recognizes a CNS tau protein rather than a tau protein, and wherein the antibody specifically recognizes a protein encoded by tau The amino acid sequence encoded by the amino acid sequence encoded by exon 4 of the gene and the amino acid sequence encoded by the exon 5 thereof serves as an epitope.

Monoclonal antibodies directed against Tau pS422 are described, for example, in EP 1 876 185. Multi-strain antibodies against Tau pS422 are commercially available (e.g., ProSci Inc. and Biosource International).

Phosphorylation of tau protein at Ser202/Thr205 is reported in WO 2006/055178, which involves contacting a sample containing tau protein with an antibody or antigen-binding fragment that binds to an amyloid β-derived diffusible ligand. Thereby inhibiting the phosphorylation of the tau protein at Ser202/Thr205.

Antibody preparations that specifically bind to tau phosphorylated at tyr394 and/or tyr310 are reported in WO 2007/019273. Animal studies using vaccinated Tau fragment 379-408 [P-Ser396, 404] to vaccinate Tau transgenic mice are mentioned in Asuni, A. A. et al., J. Neuroscience 27 (2007) 9115-9129.

EP 2 009 104 relates to an epitope of a Tau protein present in a phosphorylated state in a Tau protein from Alzheimer's disease PHF and the use of the epitope, which is used to specifically detect Alzheimer's An antibody to the Tau protein.

US 2008/0050383 relates to a method of treating and preventing Alzheimer's disease or other Tau protein disease in an individual by administering a Tau protein fragment.

An isolated synthetic or recombinant polypeptide or peptide is reported in WO 2010/037135, the polypeptide or peptide comprising a first domain comprising or consisting of a ligand of the blood brain barrier (BBB) receptor or equivalent And a second domain comprising or consisting of an enzyme or composition that slows the rate of aggregation of protein aggregates, inhibits the formation or reversal of protein aggregates, decomposes or dissolves protein aggregates. An antibody, in particular a monoclonal antibody or a functional part thereof, which is capable of recognizing and binding tau protein in vitro and/or in vivo, is reported in WO 2010/115843.

Monoclonal antibodies or fragments thereof which specifically bind to tau oligomers and do not bind soluble tau or tau fibrils are reported in WO 2011 026031, which are suitable for the treatment of tauopathy, such as Alzheimer's disease, progressive Nuclear paralysis and cortical basal ganglia degeneration. An isolated antibody that specifically binds to a human tau protein that is phosphorylated at one or more of Ser (238) and Thr (245) is reported in WO 2011/053565.

Antibodies that specifically bind to phosphorylation epitopes on mammalian Tau proteins are reported in WO 2012/045882, which are useful for the treatment of neurodegenerative diseases such as tauopathy and for the treatment or alleviation of cognitive deficits. Human monoclonal anti-tau antibodies or their tau-binding fragments are reported in WO 2012/049570. WO 2012/106363 reports a method of preventing or treating Alzheimer's disease or other Tau protein disease in an individual comprising administering to a human in need of treatment for Alzheimer's disease or other tauopathy These antibodies are specific for an abnormal form of Tau protein which does not exhibit binding and/or reactivity to normal Tau protein and is effective under conditions and amounts for preventing or treating Alzheimer's disease or other tauopathy Cast.

Antibodies exhibiting reactivity with aggregated tau and substantially non-reactive with non-aggregated Tau are reported in WO 2012/149365, wherein the aggregated tau comprises at least two at one or more cysteine residues directly or via a linker to each other Cross-linked tau protein.

A composition suitable for the treatment of a Tau protein disease such as Alzheimer's disease, which comprises an antibody that binds to Tau, and a specific phosphate at a specific position, is reported in WO 2010/142423 The Tau and its fragments specifically bind to the phosphorylated serine-modified compound and carrier.

Antibodies recognizing phosphorylated polypeptides are reported in EP 1 876 185 A. Humanized tau antibodies are reported in WO 2013/151762. A novel chicken monoclonal antibody against human phosphorylated tau and its use are reported in WO 2014/016737.

The delivery of pharmaceutical agents via the human insulin receptor is reported in WO 2004/050016. Manich, G. et al., (Eur. J Pharm. Sci. 49 (2013) 556-564) reported trans-endocytosis studies of anti-transferrin receptor antibodies using Fab' load across the blood-brain barrier in mice. Dufes, C. et al. report the transferrin and transferrin receptors for targeted delivery of therapeutic agents to brain and cancer cells (Ther. Deliv. 4 (2013) 629-640). Feng, J-M. et al., Neurometh. 45 (2010) 15-34 report receptor-mediated drug cross-BBB transmission. Pardridge, W. et al. reported reengineering biopharmaceuticals for delivery to the brain along with the Trojan horse (Bioconjug. Chem. 19 (2008) 1327-1338). Multi-specific non-covalent complex targeted delivery therapy is reported in WO 2004/045642. Ferrari, A. et al. report the paired helical fibrillar-like tau filaments in β-amyloid-induced tissue culture (J. Biol. Chem. 278 (2003) 40162-40168). Deters, N. et al., Biochem. Biophys. Res. 379 (2009) 400-405) report substrate-specific reduction of PP2A activity to augment tau lesions.

The present invention provides an anti-human Tau (pS422) antibody brain shuttle construct and a method of using the same.

Covalent conjugates of anti-human Tau (pS422) antibodies to brain shuttle modules, such as monovalent anti-human transferrin receptor antibodies or antibody fragments, have been found to reduce Tau-related lesions compared to unconjugated brain shuttles The module's anti-human Tau (pS422) antibody is less efficient. In addition, brain shuttle conjugates appear to have (neuro) toxicity. Without being bound by this theory, this may be due to the mode of action of the antibody and the cellular localization of the antibody target.

Therefore, non-covalent complexes as reported herein are reported for use in functional antibiotics. Tau (pS422) antibodies are transmitted through the blood-brain barrier.

The construct as reported herein comprises a blood-brain barrier shuttle module (BBB-shuttle module) having a first binding specificity for a hapten and a second binding to a blood brain barrier receptor (BBBR) Specific bispecific antibodies. Such BBB-shuttle modules recognize cell surface targets (such as TfR, LRP or other targets = BBBR) that are capable of transcytosis on the blood-brain barrier and simultaneously bind to the haptenated payload.

No more requirements regarding binding valence, antibody type, BBBR binding affinity have been found to be met.

It has also been found that trans-endocytosis of the haptenation payload is mediated by the release of endothelial cells from the blood-brain barrier (i.e., in complex form) of the bispecific antibody-based shuttle module as reported herein. The fact is that the haptenated payload of the shuttle-based sub-module based on the bispecific antibody is combined/non-covalently bound to the bispecific antibody-based shuttle module, i) from the BBB intracellular bispecific antibody The shuttle module is released, ie, internalized in the intracellular vesicle system, ii) separated from the shuttle module, and iii) subsequently excreted from the BBB cells into the brain (leaving bispecific in the BBB cells) Sexual antibodies).

The bispecific antibody-based shuttle module as reported herein is highly variable with respect to BBBR binding specific valency and BBBR binding specific affinity. At the same time, it enables the payload to be released from the shuttle module.

One aspect as reported herein is i) a non-covalent complex that specifically binds to a haptenated antibody of human Tau (pS422) and ii) an anti-blood brain barrier receptor/hapten bispecific antibody.

The anti-blood-brain barrier receptor/hapten bispecific antibody is a bispecific antibody comprising a specific binding to a hapten (eg, a hapten that specifically binds to a haptenated antibody of human Tau (pS422)) A binding specificity, and a second binding specificity that specifically binds to the blood brain barrier receptor.

One aspect as reported herein is that i) specifically binds to human Tau (pS422) and half A bispecific antibody to the antigen and ii) a non-covalent complex of the haptenized anti-blood brain barrier receptor antibody.

An antibody that specifically binds to human Tau (pS422) and a hapten is a bispecific antibody comprising a first binding specifically binding to a hapten (eg, a haptenated antibody that specifically binds to the blood brain barrier receptor) Specificity, and specific binding to the second binding specificity of human Tau (pS422).

One aspect as reported herein is a non-covalent complex comprising i) a haptenated antibody that specifically binds to human Tau (pS422) and ii) a bispecific antibody that specifically binds to human Tau (pS422) a first binding specificity of a hapten that specifically binds to a haptenated antibody and a second binding specificity that specifically binds to a blood brain barrier receptor, wherein the hapten specifically binds to human Tau (pS422) The antibody specifically binds by the first binding specificity of the bispecific antibody.

One aspect as reported herein is a non-covalent complex comprising i) a haptenated antibody that specifically binds to the blood brain barrier receptor, and ii) a bispecific antibody that specifically binds to human Tau The first binding specificity (pS422) binds specifically to the second binding specificity of the hapten of the haptenized antibody that specifically binds to the blood brain barrier receptor, wherein the specific binding to the blood brain barrier receptor is half The antigenized antibody specifically binds by the second binding specificity of the bispecific antibody.

One aspect as reported herein is the use of a non-covalent complex that specifically binds to a haptenated antibody of human Tau (pS422) and an anti-blood brain barrier receptor/hapten bispecific antibody, which is used Antibodies that specifically bind to human Tau (pS422) are transmitted through the blood brain barrier.

One aspect as reported herein is the use of a non-covalent complex of a bispecific antibody that specifically binds to human Tau (pS422) and a hapten and a haptenized anti-blood brain barrier receptor antibody, which is used Antibodies that specifically bind to human Tau (pS422) are transmitted through the blood brain barrier.

One aspect as reported herein is the use of a non-covalent complex comprising a haptenated antibody and a bispecific antibody that specifically binds to human Tau (pS422), which specifically binds to human Tau (pS422) a first binding specificity of a hapten that specifically binds to a haptenated antibody and a second binding specificity that specifically binds to a blood brain barrier receptor, wherein the haptenated antibody specifically binds to human Tau (pS422) The non-covalent complex is used to transport antibodies that specifically bind to human Tau (pS422) across the blood brain barrier by first binding specific binding of a bispecific antibody.

One aspect as reported herein is the use of a non-covalent complex comprising a haptenated antibody and a bispecific antibody that specifically binds to a blood brain barrier receptor, the bispecific antibody having specific binding to humans The first binding specificity and specificity of Tau (pS422) binds to the second binding specificity of the hapten of a haptenized antibody that specifically binds to the blood brain barrier receptor, wherein it specifically binds to the blood brain barrier receptor A haptenated antibody specifically binds by a first binding specificity of a bispecific antibody for transporting an antibody that specifically binds to human Tau (pS422) across the blood brain barrier.

In one embodiment, the haptenation anti-system is selected from the group consisting of biotinylated antibodies, theophyllined antibodies, digoxigenin antibodies, carboborated antibodies, luciferylated antibodies, helical antibodies, and bromine Deoxyuridine antibody. In a preferred embodiment, the haptenated antibody is a biotinylated antibody or a digoxigenin antibody.

In one embodiment, the haptenated anti-system that specifically binds to human Tau (pS422) is selected from the group consisting of a biotinylated antibody that specifically binds to human Tau (pS422), specifically binds to human Tau ( a theophylline antibody of pS422), a digoxigenin antibody that specifically binds to human Tau (pS422), a carboborated antibody that specifically binds to human Tau (pS422), and a fluorescein that specifically binds to human Tau (pS422) A phototinylated antibody, a spirospecific antibody that specifically binds to human Tau (pS422), and a bromodeoxyuridine antibody that specifically binds to human Tau (pS422). In a preferred embodiment, the haptenated antibody that specifically binds to human Tau (pS422) is biotinylated specifically binding to human Tau (pS422) An antibody or a digoxigenin antibody that specifically binds to human Tau (pS422).

In one embodiment, the haptenated anti-system that specifically binds to the blood-brain barrier receptor is selected from the group consisting of biotinylated antibodies that specifically bind to the blood-brain barrier receptor, specifically bind to the blood-brain barrier. a theophylline antibody of the receptor, a digoxigenin antibody that specifically binds to the blood-brain barrier receptor, a carboplatin antibody that specifically binds to the blood-brain barrier receptor, and a fluorescein that specifically binds to the blood-brain barrier receptor A phototinylated antibody, a helical antibody that specifically binds to a blood-brain barrier receptor, and a bromodeoxyuridine antibody that specifically binds to a blood-brain barrier receptor. In a preferred embodiment, the haptenated antibody that specifically binds to the blood-brain barrier receptor is a biotinylated antibody that specifically binds to the blood-brain barrier receptor or specifically binds to the blood-brain barrier receptor. Xinhua antibody.

In one embodiment, the blood-brain barrier receptor system is selected from the group consisting of: transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF receptor), low density lipoprotein receptor associated Protein 8 (LRP8), low density lipoprotein receptor associated protein 1 (LRP1) and heparin bind epidermal growth factor-like growth factor (HB-EGF). In a preferred embodiment, the blood brain barrier receptor is a transferrin receptor.

In one embodiment, the bispecific antibody is a full length antibody comprising two binding sites.

In one embodiment, the bispecific antibody is a full length antibody that has been fused to one or two scFv or scFab or CrossFab or scCrossFab and comprises three or four binding sites.

In one embodiment, the bispecific antibody is an antibody fragment. In one embodiment, the antibody fragment is selected from the group consisting of F(ab')2 and a bifunctional antibody.

In one embodiment, the bispecific antibody is a humanized antibody or a human antibody.

In one embodiment, the bispecific antibody does not contain an effector function. In one embodiment, the bispecific antibody does not have a functional Fc region. In one embodiment, the bispecific antibody does not have an Fc region. In one embodiment, the bispecific antibody has a mutation The Fc region of the human IgGl subclass of L234A, L235A and P329G, wherein these positions are determined according to the Fc region number of Kabat (Kabat EU Index). In one embodiment, the bispecific antibody has an Fc region comprising a human IgG4 subclass of the mutations S228P, L235E and P329G, wherein the positions are determined according to the Fc region number of Kabat (Kabat EU Index).

In one embodiment, the bispecific antibody comprises a) a binding site for a hapten of a haptenated antibody and a binding site for a blood brain barrier receptor, or b) two for a haptenated antibody a binding site for a hapten and a binding site for a blood brain barrier receptor, or c) a binding site for a hapten of a haptenated antibody and two binding sites for a blood brain barrier receptor, or d) two binding sites for the hapten of the haptenated antibody and two binding sites for the blood brain barrier receptor.

In one embodiment, the bispecific antibody comprises a) a binding site for a hapten that specifically binds to a haptenated antibody of human Tau (pS422) and a binding site for a blood brain barrier receptor, or b) two binding sites for a hapten that specifically binds to a haptenized antibody of human Tau (pS422) and a binding site for a blood brain barrier receptor, or c) a specific binding to human Tau (pS422) a binding site for a hapten of a haptenated antibody and two binding sites for a blood brain barrier receptor, or d) two for a haptenated antibody that specifically binds to human Tau (pS422) The binding site of the hapten and two binding sites for the blood brain barrier receptor.

In one embodiment, the bispecific antibody comprises a) a binding site for a hapten that specifically binds to a haptenated antibody of the blood brain barrier receptor and a binding site for human Tau (pS422), or b) two semi-antibodies against haptenated antibodies that specifically bind to the blood-brain barrier receptor The original binding site and a binding site for human Tau (pS422), or c) a binding site for a hapten that specifically binds to the hemantigenic antibody of the blood-brain barrier receptor and two for human Tau a binding site for (pS422), or d) two binding sites for a hapten that specifically binds to a haptenized antibody of the blood brain barrier receptor and two binding sites for human Tau (pS422).

In cases b) and c) of the foregoing examples, one of the heavy chains of the bispecific antibody comprises a purine mutation and each of the other strands comprises a purine mutation.

In a preferred embodiment, the bispecific antibody comprises two binding sites for a hapten that specifically binds to a haptenated antibody of human Tau (pS422) and two binding sites for a blood brain barrier receptor .

In a preferred embodiment, the bispecific antibody comprises two binding sites for a hapten that specifically binds to a haptenized antibody of the blood brain barrier receptor and two binding sites for human Tau (pS422) .

In one embodiment, the bispecific antibody has two binding specificities (two anti-hapten binding specificities) and specificity for a hapten that specifically binds to a haptenized antibody that specifically binds to human Tau (pS422) Sexual binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor-related protein 8 binding specificity) Two combinations of specificity.

In one embodiment, the bispecific antibody specifically binds to a protein associated with (human) transferrin receptor (two anti-hapten binding specificity) or low density lipoprotein receptor 8 (two anti-haptens) Binding specificity) The binding specificity and specificity of the hapten of a specifically bound haptenated antibody binds to both binding specificities of human Tau (pS422).

In one embodiment, the binding specificity of digoxin that specifically binds to a digoxigenin antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, Including (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 65, (b) a heavy chain CDR2 comprising the amino acid sequence SEQ ID NO: 66, (c) comprising an amino acid sequence SEQ ID NO: 67 heavy chain CDR3, (d) comprising the amino acid sequence SEQ ID NO: 69 of the light chain CDR1, (e) comprising the amino acid sequence SEQ ID NO: 70 of the light chain CDR2 and (f) comprising an amine The light chain CDR3 of the base acid sequence of SEQ ID NO:71.

In one embodiment, the binding specificity of digoxin that specifically binds to a digoxigenin antibody that specifically binds to human Tau (pS422) is humanized binding specificity.

In one embodiment, the binding specificity of digoxin that specifically binds to a digoxigenin antibody that specifically binds to human Tau (pS422) comprises a CDR as described in the above Examples and a human framework of the acceptor (eg, human immunity) Globulin framework or human common framework).

In one embodiment, the binding specificity of digoxin that specifically binds to a digoxigenin antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, Included in that (a) the heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 73, (b) the heavy chain CDR2 comprising the amino acid sequence SEQ ID NO: 74, (c) comprising the amino acid sequence SEQ ID NO: 75 The heavy chain CDR3, (d) comprises the amino acid sequence SEQ ID NO: 77 of the light chain CDR1, (e) comprises the amino acid sequence SEQ ID NO: 78 of the light chain CDR2 and (f) comprises the amino acid sequence SEQ ID NO: 79 light chain CDR3.

In one embodiment, the binding specificity of digoxin that specifically binds to a digoxigenin antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, Included with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence SEQ ID NO: 68 or 76 Heavy chain variable domain (VH) sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, conservative substitutions, insertions or deletions, but the anti-digoxigenin antibody comprising the sequence retains the ability to bind to digoxin. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 68 or 76 have been substituted, inserted, and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). Digoxin binding specificity comprises SEQ ID NO: 68 or 76, as appropriate A VH sequence, including post-translational modifications of the sequence.

In one embodiment, the binding specificity of digoxin that specifically binds to a digoxigenin antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, Further comprising a sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence SEQ ID NO: 72 or 80 Sexual light chain variable domain (VL) sequence. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, conservative substitutions, insertions or deletions, but the anti-digoxigenin antibody comprising the sequence retains the ability to bind to digoxin. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 72 or 80 have been substituted, inserted, and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). Optionally, the digoxigenin binding specificity comprises the VL sequence of SEQ ID NO: 72 or 80, including post-translational modifications of the sequence.

In one embodiment the bispecific antibody comprises a first binding specificity (antibiotic binding specificity; anti-BI binding specificity) of biotin that specifically binds to a biotinylated antibody that specifically binds to human Tau (pS422) And specific binding to (human) transferrin receptor (anti-(human) transferrin receptor binding specificity; anti-(h)TfR binding specificity) or low-density lipoprotein receptor-related protein 8 (anti- Low binding lipoprotein receptor-related protein 8 binding specificity; anti-LRP8 binding specificity) second binding specificity.

In one embodiment, the bispecific antibody has two binding specificities (two avidin binding specificities) and specificity for biotin that specifically binds to a biotinylated antibody that specifically binds to human Tau (pS422) Sexual binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor-related protein 8 binding specificity) Two binding specificities of sex).

In one embodiment, the binding specificity of biotin that specifically binds to a biotinylated antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain A variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 81, (b) a heavy chain CDR2 comprising the amino acid sequence SEQ ID NO: 82, (c) comprising an amino acid The heavy chain CDR3 of SEQ ID NO: 83, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 85, (e) comprises the light chain CDR2 of the amino acid sequence SEQ ID NO: 86, and (f ) a light chain CDR3 comprising the amino acid sequence SEQ ID NO:87.

In one embodiment, the binding specificity of biotin that specifically binds to a biotinylated antibody that specifically binds to human Tau (pS422) is humanized binding specificity.

In one embodiment, the binding specificity of biotin that specifically binds to a biotinylated antibody that specifically binds to human Tau (pS422) comprises a CDR as described in the above Examples and a human framework of the acceptor (eg, human immunoglobulin) Architecture or human common architecture).

In one embodiment, the binding specificity of biotin that specifically binds to a biotinylated antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, which comprises ( a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 89, (b) a heavy chain CDR2 comprising the amino acid sequence SEQ ID NO: 90, (c) comprising the amino acid sequence SEQ ID NO: 91 The CDR3, (d) comprises the amino acid sequence SEQ ID NO: 93, the light chain CDR1, (e) comprises the amino acid sequence SEQ ID NO: 94, the light chain CDR2, and (f) comprises the amino acid sequence SEQ ID NO: 95 light chain CDR3.

In one embodiment, the binding specificity of biotin that specifically binds to a biotinylated antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, which comprises Amino acid sequence SEQ ID NO: 84 or 92 has a weight of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Chain variable domain (VH) sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, conservative substitutions, insertions or deletions, but the anti-biotin antibody comprising the sequence retains the ability to bind to biotin. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 84 or 92 have been substituted, inserted and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). The biotin binding specificity comprises, as the case may be, the VH sequence of SEQ ID NO: 84 or 92, including post-translational modifications of the sequence.

In one embodiment, the binding specificity of biotin that specifically binds to a biotinylated antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, which additionally comprises Having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence SEQ ID NO: 88 or 96 Light chain variable domain (VL). In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, conservative substitutions, insertions or deletions, but the anti-biotin antibody comprising the sequence retains the ability to bind to biotin. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 88 or 96 have been substituted, inserted, and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). Depending on the case, the biotin binding specificity comprises the VL sequence of SEQ ID NO: 88 or 96, which includes post-translational modifications of the sequence.

In one embodiment, the bispecific antibody comprises a first binding specificity (an anti-theophylline binding specificity; an anti-theoO binding specificity) of a theophylline that specifically binds to a theophylline antibody that specifically binds to human Tau (pS422) And specific binding to (human) transferrin receptor (anti-(human) transferrin receptor binding specificity; anti-(h)TfR binding specificity) or low-density lipoprotein receptor-related protein 8 ( Second binding specificity against low density lipoprotein receptor associated protein 8 binding specificity; anti-LRP8 binding specificity).

In one embodiment, the bispecific antibody has two binding specificities (two anti-theophylline binding specificities) and specificity for theophylline that specifically binds to the theophylline antibody that specifically binds to human Tau (pS422) Sexual binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor-related protein 8 binding specificity) Two binding specificities of sex).

In one embodiment, the binding specificity of a theophylline that specifically binds to a theophylline antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, which comprises (a a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 97, (b) a heavy chain CDR2 comprising the amino acid sequence SEQ ID NO: 98, (c) a heavy chain comprising the amino acid sequence SEQ ID NO: 99 CDR3, (d) comprises the amino acid sequence SEQ ID NO: 101 of the light chain CDR1, (e) comprises the amino acid sequence SEQ ID NO: 102, the light chain CDR2, and (f) comprises the amino acid sequence SEQ ID NO : 103 light chain CDR3.

In one embodiment, the binding specificity of a theophylline that specifically binds to a theophylline antibody that specifically binds to human Tau (pS422) is a humanized binding specificity.

In one embodiment, the binding specificity of a theophylline that specifically binds to a theophylline antibody that specifically binds to human Tau (pS422) comprises a CDR according to the above examples and a human framework of the acceptor (eg, human immunoglobulin) Architecture or human common architecture).

In one embodiment, the binding specificity of a theophylline that specifically binds to a theophylline antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, which comprises (a a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 105, (b) a heavy chain CDR2 comprising the amino acid sequence SEQ ID NO: 106, (c) a heavy chain comprising the amino acid sequence SEQ ID NO: 107 CDR3, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 109, (e) comprises the light chain CDR2 of the amino acid sequence SEQ ID NO: 110, and (f) comprises the amino acid sequence SEQ ID NO : 111 light chain CDR3.

In one embodiment, the binding specificity of a theophylline that specifically binds to a theophylline antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, which comprises Amino acid sequence SEQ ID NO: 100 or 108 has a weight of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Chain variable domain (VH) sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence contains a substitution ( Such as conservative substitutions), insertions or deletions, but The anti-theophylline antibody of this sequence retains the ability to bind to theophylline. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 100 or 108 have been substituted, inserted and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). Optionally, the theophylline binding specificity comprises the VH sequence of SEQ ID NO: 100 or 108, including post-translational modifications of the sequence.

In one embodiment, the binding specificity of a theophylline that specifically binds to a theophylline antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair additionally comprising an amine Base acid sequence SEQ ID NO: 104 or 112 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the light chain Variable domain (VL). In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence contains a substitution ( For example, conservative substitutions, insertions or deletions, but the anti-theophylline antibody comprising the sequence retains the ability to bind to theophylline. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 104 or 112 have been substituted, inserted, and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). Optionally, the theophylline binding specificity comprises the VL sequence of SEQ ID NO: 104 or 112, including post-translational modifications of the sequence.

In one embodiment, the bispecific antibody comprises a first binding specificity (anti-luciferin binding specificity) of luciferin that specifically binds to a luciferylated antibody that specifically binds to human Tau (pS422); FLUO binding specificity and specific binding to (human) transferrin receptor (anti-(human) transferrin receptor binding specificity; anti-(h)TfR binding specificity) or low density lipoprotein receptor Second binding specificity of protein 8 (anti-low density lipoprotein receptor associated protein 8 binding specificity; anti-LRP8 binding specificity).

In one embodiment, the bispecific antibody has two binding specificities for luciferin that specifically binds to a luciferylated antibody that specifically binds to human Tau (pS422) (two anti-luciferin binding specificities) And specific binding to (human) transferrin receptor (two anti-human Two binding specificities of the class of transferrin receptor binding specificity or low density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor-related protein 8 binding specificity).

In one embodiment, the binding specificity of a luciferin that specifically binds to a luciferylated antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 113, (b) comprising a heavy chain CDR2 of the amino acid sequence SEQ ID NO: 114, (c) comprising an amino acid sequence of SEQ ID NO: 115 The heavy chain CDR3, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 117, (e) comprises the light chain CDR2 of the amino acid sequence SEQ ID NO: 118, and (f) comprises the amino acid sequence SEQ ID NO: 119 light chain CDR3.

In one embodiment, the binding specificity of a luciferin that specifically binds to a luciferylated antibody that specifically binds to human Tau (pS422) is a humanized binding specificity.

In one embodiment, the binding specificity of a luciferin that specifically binds to a luciferylated antibody that specifically binds to human Tau (pS422) comprises a CDR as described in the above Examples and a human framework of the acceptor (eg, human immunity) Globulin framework or human common framework).

In one embodiment, the binding specificity of a luciferin that specifically binds to a luciferylated antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, Further comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence SEQ ID NO:116 Heavy chain variable domain (VH) sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, conservative substitutions, insertions or deletions, but the anti-luciferin antibody comprising the sequence retains the ability to bind to luciferin. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 116 have been substituted, inserted, and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). Depending on the case, the luciferin binding specificity comprises the VH sequence of SEQ ID NO: 116, including post-translational modifications of the sequence.

In one embodiment, the binding specificity of a luciferin that specifically binds to a luciferylated antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable domain pair, Further comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence SEQ ID NO: 120 Light chain variable domain (VL) sequence. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, conservative substitutions, insertions or deletions, but the anti-luciferin antibody comprising the sequence retains the ability to bind to luciferin. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 120 have been substituted, inserted, and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). Depending on the case, the luciferin binding specificity comprises the VL sequence of SEQ ID NO: 120, including post-translational modifications of the sequence.

In one embodiment, the bispecific antibody comprises a first binding specificity (bromodeoxyuridine binding specificity; anti-BrdU binding specificity) and specific binding to a net payload of bromodeoxyuridine (Human) transferrin receptor (anti-(human) transferrin receptor binding specificity; anti-(h)TfR binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor) The second binding specificity of the relevant protein 8 binding specificity; anti-LRP8 binding specificity).

In one embodiment, the bispecific antibody has two binding specificities (two anti-bromodeoxyuridine binding specificities) that specifically bind to the payload of bromodeoxyuridine and specific binding to (human) Two combinations of transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor-related protein 8 binding specificity) Specificity.

In one embodiment, the binding specificity of a bromodeoxyuridine that specifically binds to a bromodeoxyuridine antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable Domain pair comprising (a) a heavy chain comprising the amino acid sequence SEQ ID NO: 121 CDR1, (b) comprises the heavy chain CDR2 of the amino acid sequence SEQ ID NO: 123, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 125, and (d) comprises the amino acid sequence SEQ ID NO: The light chain CDR1 of 126, (e) comprises the light chain CDR2 of the amino acid sequence SEQ ID NO: 127, and (f) comprises the light chain CDR3 of the amino acid sequence SEQ ID NO: 128.

In one embodiment, the binding specificity of bromodeoxyuridine that specifically binds to a bromodeoxyuridine antibody that specifically binds to human Tau (pS422) is humanized binding specificity.

In one embodiment, the binding specificity of bromodeoxyuridine that specifically binds to a bromodeoxyuridine antibody that specifically binds to human Tau (pS422) comprises a CDR and acceptor human framework as in the above examples. (eg human immunoglobulin framework or human common framework).

In one embodiment, the binding specificity of a bromodeoxyuridine that specifically binds to a bromodeoxyuridine antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable A domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 121 or 122, (b) a heavy chain CDR2 comprising the amino acid sequence SEQ ID NO: 123 or 124, (c) comprising an amine a heavy chain CDR3 of SEQ ID NO: 126, (d) comprising the light chain CDR1 of the amino acid sequence SEQ ID NO: 126, (e) comprising the light chain CDR2 of the amino acid sequence SEQ ID NO: 127, and (f) a light chain CDR3 comprising the amino acid sequence SEQ ID NO:128.

In one embodiment, the binding specificity of a bromodeoxyuridine that specifically binds to a bromodeoxyuridine antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable a domain pair comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 with the amino acid sequence SEQ ID NO: 129 or 131 % sequence-consistent heavy chain variable domain (VH) sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( Such as conservative substitutions), insertions or deletions, However, the anti-bromodeoxyuridine antibody comprising the sequence retains the ability to bind to bromodeoxyuridine. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 129 or 131 have been substituted, inserted and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). Depending on the case, the bromodeoxyuridine binding specifically comprises the VH sequence of SEQ ID NO: 129 or 131, including post-translational modifications of the sequence.

In one embodiment, the binding specificity of a bromodeoxyuridine that specifically binds to a bromodeoxyuridine antibody that specifically binds to human Tau (pS422) is an antibody heavy chain variable domain and an antibody light chain variable a domain pair additionally comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or with the amino acid sequence SEQ ID NO: 130 or 132 or A light chain variable domain (VL) sequence of 100% sequence identity. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, conservative substitutions, insertions or deletions, but the anti-bromo-deoxyuridine antibody comprising the sequence retains the ability to bind to bromodeoxyuridine. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 130 or 132 have been substituted, inserted and/or deleted. In certain embodiments, a substitution, insertion or deletion occurs in the outer region of the CDR (ie, in the FR). Depending on the case, the bromodeoxyuridine binding specifically comprises the VL sequence of SEQ ID NO: 130 or 132, including post-translational modifications of the sequence.

In one embodiment, a haptenized antibody that specifically binds to human Tau (pS422) comprises a linking group between a hapten and an antibody that specifically binds to human Tau (pS422). In one embodiment, the linking group is a peptide linking group. In one embodiment, the linking group is a chemical linking group (non-peptide linking group).

In one embodiment, the antibody that specifically binds to human Tau (pS422) is a full length antibody.

In one embodiment, an antibody that specifically binds to human Tau (pS422) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to the full length at 1 μg/mL Human Tau (SEQ ID NO: 01), and/or Iii) full length human Tau (SEQ ID NO: 02), and/or iv) that specifically binds to phosphorylation at position 422, and specifically binds to phosphorylation at serine at position 422 Aggregates of human Tau (SEQ ID NO: 02), and/or v) specifically bind to human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A.

Antibodies specifically binding to human Tau (pS422) as reported herein show human Tau phosphorylation at the position of sarminic acid at position 422, with respect to the selectivity of unphosphorylated wild-type human Tau and Tau mutant S422A. Unphosphorylated wild-type human Tau and Tau mutant S422A do not bind at all or have lower affinity, respectively.

In one embodiment, an antibody that specifically binds to human Tau (pS422) comprises a) a heavy chain variable domain, HVR of SEQ ID NOs: 08, 18, and 10, or b) in a heavy chain variable domain , HVR of SEQ ID NOS: 08, 09 and 10.

In one embodiment, the antibody that specifically binds to human Tau (pS422) additionally comprises a) in the light chain variable domain, the HVR of SEQ ID NOs: 13, 14 and 15, or b) is variable in the light chain In the domain, HVRs of SEQ ID NOS: 12, 05 and 15.

In one embodiment, an antibody that specifically binds to human Tau (pS422) comprises a) an HVR of SEQ ID NOs: 08, 18, and 10 in the heavy chain variable domain, and in the light chain variable domain , HVR of SEQ ID NOS: 13, 14 and 15, or b) HVR of SEQ ID NOS: 08, 09 and 10 in the heavy chain variable domain, and SEQ ID NO in the light chain variable domain HVR of 12, 05 and 15, or c) HVR of SEQ ID NOS: 08, 09 and 10 in the heavy chain variable domain, and SEQ ID NO: 13, 14 in the light chain variable domain And 15 HVR.

In one embodiment, the antibody that specifically binds to human Tau (pS422) comprises a) a heavy chain variable domain of SEQ ID NO: 20 and a light chain variable domain of SEQ ID NO: Or b) the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 16, or c) the heavy chain variable domain of SEQ ID NO: 19 and the light chain of SEQ ID NO: The variable domain, or d) the heavy chain variable domain of SEQ ID NO: 21 and the light chain variable domain of SEQ ID NO: 17.

In one embodiment, the non-covalent complex is used to treat Alzheimer's disease.

In one embodiment, both antibodies in the complex are functionally functionally silent. In one embodiment, neither antibody of the complex has an effector function.

In one embodiment, an antibody that specifically binds to human Tau (pS422) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to the full length at 1 μg/mL Human Tau (SEQ ID NO: 01), and/or iii) specifically binds to full-length human Tau (SEQ ID NO: 02) phosphorylated at position 422, and/or iv) specifically binds Aggregates of human Tau (SEQ ID NO: 02) phosphorylated at the serine acid at position 422.

In one embodiment, specific binding to a human Tau (pS422) the antibody has the following EC ₅₀ values of a) an amino acid sequence of SEQ ID NO: Human Tau (pS422) fragment of 03, 6ng / mL or 6ng / mL or less And/or b) full length human Tau (pS422) having the amino acid sequence SEQ ID NO: 02, 4.5 ng/mL or 4.5 ng/mL or less, and/or c) having the amino acid sequence SEQ ID NO: 02 Human Tau (pS422) aggregate, 30 ng/mL or less, and/or d) human Tau with amino acid sequence SEQ ID NO: 01 and amino acid mutation S422A, 125 ng/mL or 125 ng/ Below mL.

In one embodiment, an antibody that specifically binds to human Tau (pS422) (SEQ ID NO: 02) does not bind to human Tau (SEQ ID NO: 01).

In one embodiment, the antibody that specifically binds to human Tau (pS422) is a monoclonal antibody.

In one embodiment, the antibody that specifically binds to human Tau (pS422) is an antibody fragment that binds to human Tau (pS422) and i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, And/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) fully binds to full length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 01) ID NO: 02), and/or iv) an aggregate that specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422, and/or v) specifically binds to A full length human Tau having the amino acid sequence of SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) having a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or 6 ng / mL of the following ₅₀ values EC, and / or vii) having the amino acid sequence of SEQ ID NO: human Tau (pS422) with a fragment of the 02 ₅₀ value of 4.5 ng / mL or 4.5 ng / mL or less of the EC, and / or Viii) having an EC ₅₀ value of 30 ng/mL or less, and/or ix) pairs of human Tau (pS422) aggregates having the amino acid sequence SEQ ID NO: 02, and having the amino acid sequence SEQ ID NO: 01 and has an amine S422A mutant of human Tau acid having ₅₀ values 125ng / mL or 125ng / mL or less of the EC.

In one embodiment, the antibody that specifically binds to human Tau (pS422) is a) a full length antibody to human subclass IgG1, or b) a full length antibody to human subclass IgG4, or c) has mutations L234A, L235A, and P329G Full length resistance of human subclass IgG1 , d) full-length antibody to human subclass IgG4 with mutations S228P, L235E and P329G, e) with mutations L234A, L235A and P329G in two heavy chains and mutations T366W and S354C in one heavy chain and another A full-length antibody of human subclass IgG1 having the mutations T366S, L368A, Y407V and Y349C, or f) having mutations S228P and P329G in two heavy chains and having mutations T366W and S354C in one heavy chain and in another heavy chain A full-length antibody of human subclass IgG4 having mutations T366S, L368A, Y407V and Y349C.

In one embodiment, antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) variable domain comprises SEQ ID NO: 08, SEQ ID NO: 18 and HVR of SEQ ID NO: 10, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amine acid residue, and iii) the constant region comprises an amine group Acidic changes L234A, L235A and P329G, b) comprise two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain comprises SEQ ID NO: 13, SEQ ID NO: 14 And HVR of SEQ ID NO: 15, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, And/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) fully binds to full length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 01) ID NO: 02), and/or iv) an aggregate that specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422, and/or v) specifically binds to Amino acid a full length human Tau having SEQ ID NO: 01 and having amino acid mutation S422A, and/or vi) having a human Tau (pS422) fragment having amino acid sequence SEQ ID NO: 03 of 6 ng/mL or less the EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL or less of EC ₅₀ values, and / or viii) to A human Tau (pS422) aggregate having an amino acid sequence of SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has S422A mutation of amino acid ₅₀ with human Tau value of 125ng / mL or 125ng / mL or less of the EC.

In one embodiment, antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) variable domain comprises SEQ ID NO: 08, SEQ ID NO: 09 and HVR of SEQ ID NO: 10, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amine acid residue, and iii) the constant region comprises an amine group Acid changes L234A, L235A and P329G, b) comprise two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain comprises SEQ ID NO: 12, SEQ ID NO: 05 And HVR of SEQ ID NO: 15, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, And/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) fully binds to full length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 01) ID NO: 02), and/or iv) an aggregate that specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422, and/or v) specifically binds to Amino acid a full length human Tau having the sequence SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) having a human Tau (pS422) fragment having the amino acid sequence of SEQ ID NO: 03 of 6 ng/mL or less the EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL or less of EC ₅₀ values, and / or viii) to A human Tau (pS422) aggregate having an amino acid sequence of SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has S422A mutation of amino acid ₅₀ with human Tau value of 125ng / mL or 125ng / mL or less of the EC.

In one embodiment, antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) variable domain comprises SEQ ID NO: 08, SEQ ID NO: 09 and HVR of SEQ ID NO: 10, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amine acid residue, and iii) the constant region comprises an amine group Acidic changes L234A, L235A and P329G, b) comprise two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain comprises SEQ ID NO: 13, SEQ ID NO: 14 And HVR of SEQ ID NO: 15, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, And/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) fully binds to full length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 01) ID NO: 02), and/or iv) an aggregate that specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422, and/or v) specifically binds to Amino acid a full length human Tau having SEQ ID NO: 01 and having amino acid mutation S422A, and/or vi) having a human Tau (pS422) fragment having amino acid sequence SEQ ID NO: 03 of 6 ng/mL or less the EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL or less of EC ₅₀ values, and / or viii) to A human Tau (pS422) aggregate having an amino acid sequence of SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has S422A mutation of amino acid ₅₀ with human Tau value of 125ng / mL or 125ng / mL or less of the EC.

In one embodiment, antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has an amine group Acid sequence SEQ ID NO: 20, ii) The constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amine acid residue, and iii) the constant region comprises amino acid changes L234A, L235A and P329G, b) Included are two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 17, and ii) the constant region is the human kappa light chain constant region Or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence SEQ ID NO: 03, and/or ii) does not bind to full length human Tau at 1 μg/mL (SEQ ID NO) :01), and/or iii) full-length human Tau (SEQ ID NO: 02), and/or iv) that specifically binds to the phosphorylation at serine at position 422, specifically binds at position 422 An aggregate of human Tau (SEQ ID NO: 02) phosphorylated at serine, and/or v) specifically binds to the amino acid sequence SEQ ID NO: 01 and has an amino acid mutation S422A Full-length human Tau, and / or vi) having the amino acid sequence of SEQ ID NO: Human Tau (pS422) with fragment 03 of 6ng / mL or 6ng / mL or less of EC ₅₀ values, and / or vii) to an amine acid sequence of SEQ ID NO: human Tau (pS422) with a fragment of the 02 ₅₀ value of 4.5ng / mL or 4.5ng / mL or less of the EC, and / or viii) having the amino acid sequence of SEQ ID NO: 02 of human Tau ( pS422) aggregate having a value of ₅₀ 30ng / mL or 30ng / mL or less of the EC, and / or ix) having the amino acid sequence of SEQ ID NO: 01 and having the human amino acid mutations S422A Tau with 125ng / mL or 125ng EC ₅₀ value below /mL.

In one embodiment, antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has an amine group Acid sequence SEQ ID NO: 19, ii) The constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) the constant region comprises the amino acid changes L234A, L235A and P329G, b) Included are two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 16, and ii) the constant region is the human kappa light chain constant region Or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence SEQ ID NO: 03, and/or ii) does not bind to full length human Tau at 1 μg/mL (SEQ ID NO) :01), and/or iii) full-length human Tau (SEQ ID NO: 02), and/or iv) that specifically binds to the phosphorylation at serine at position 422, specifically binds at position 422 An aggregate of human Tau (SEQ ID NO: 02) phosphorylated at serine, and/or v) specifically binds to the amino acid sequence SEQ ID NO: 01 and has an amino acid mutation S422A Full-length human Tau, and / or vi) having the amino acid sequence of SEQ ID NO: Human Tau (pS422) with fragment 03 of 6ng / mL or 6ng / mL or less of EC ₅₀ values, and / or vii) to an amine acid sequence of SEQ ID NO: human Tau (pS422) with a fragment of the 02 ₅₀ value of 4.5ng / mL or 4.5ng / mL or less of the EC, and / or viii) having the amino acid sequence of SEQ ID NO: 02 of human Tau ( pS422) aggregate having a value of ₅₀ 30ng / mL or 30ng / mL or less of the EC, and / or ix) having the amino acid sequence of SEQ ID NO: 01 and having the human amino acid mutations S422A Tau with 125ng / mL or 125ng EC ₅₀ value below /mL.

In one embodiment, antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has an amine group Acid sequence SEQ ID NO: 19, ii) The constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) the constant region comprises the amino acid changes L234A, L235A and P329G, b) Included are two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 17, and ii) the constant region is the human kappa light chain constant region Or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence SEQ ID NO: 03, and/or ii) does not bind to full length human Tau at 1 μg/mL (SEQ ID NO) :01), and/or iii) full-length human Tau (SEQ ID NO: 02), and/or iv) that specifically binds to the phosphorylation at serine at position 422, specifically binds at position 422 An aggregate of human Tau (SEQ ID NO: 02) phosphorylated at serine, and/or v) specifically binds to the amino acid sequence SEQ ID NO: 01 and has an amino acid mutation S422A Full-length human Tau, and / or vi) having the amino acid sequence of SEQ ID NO: Human Tau (pS422) with fragment 03 of 6ng / mL or 6ng / mL or less of EC ₅₀ values, and / or vii) to an amine acid sequence of SEQ ID NO: human Tau (pS422) with a fragment of the 02 ₅₀ value of 4.5ng / mL or 4.5ng / mL or less of the EC, and / or viii) having the amino acid sequence of SEQ ID NO: 02 of human Tau ( pS422) aggregate having a value of ₅₀ 30ng / mL or 30ng / mL or less of the EC, and / or ix) having the amino acid sequence of SEQ ID NO: 01 and having the human amino acid mutations S422A Tau with 125ng / mL or 125ng EC ₅₀ value below /mL.

In one embodiment, antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has an amine group Acid sequence SEQ ID NO: 21, ii) The constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) the constant region comprises the amino acid changes L234A, L235A and P329G, b) Included are two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 17, and ii) the constant region is the human kappa light chain constant region Or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence SEQ ID NO: 03, and/or ii) does not bind to full length human Tau at 1 μg/mL (SEQ ID NO) :01), and/or iii) full-length human Tau (SEQ ID NO: 02), and/or iv) that specifically binds to the phosphorylation at serine at position 422, specifically binds at position 422 An aggregate of human Tau (SEQ ID NO: 02) phosphorylated at serine, and/or v) specifically binds to the amino acid sequence SEQ ID NO: 01 and has an amino acid mutation S422A Full-length human Tau, and / or vi) having the amino acid sequence of SEQ ID NO: Human Tau (pS422) with fragment 03 of 6ng / mL or 6ng / mL or less of EC ₅₀ values, and / or vii) to an amine acid sequence of SEQ ID NO: human Tau (pS422) with a fragment of the 02 ₅₀ value of 4.5ng / mL or 4.5ng / mL or less of the EC, and / or viii) having the amino acid sequence of SEQ ID NO: 02 of human Tau ( pS422) aggregate having a value of ₅₀ 30ng / mL or 30ng / mL or less of the EC, and / or ix) having the amino acid sequence of SEQ ID NO: 01 and having the human amino acid mutations S422A Tau with 125ng / mL or 125ng EC ₅₀ value below /mL.

In a preferred embodiment of all aspects, an antibody that specifically binds to human Tau (pS422) has a proline residue at a heavy chain variable domain at positions 4, 24, and 78.

In a preferred embodiment of all aspects, an antibody that specifically binds to human Tau (pS422) has a arginine residue at the heavy chain variable domain at position 71.

One aspect as reported herein is a pharmaceutical formulation comprising a non-covalent complex as reported herein and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical formulation additionally comprises an additional therapeutic agent.

In one embodiment, the additional therapeutic agent is an anti-amyloid therapeutic. In one embodiment, the anti-amyloid therapeutic agent is an anti-human alpha synuclein antibody or an anti-Aβ antibody. In one embodiment, the anti-human alpha synuclein antibody or anti-Aβ antibody is haptenized. In one embodiment, the anti-human alpha synuclein antibody or anti-Aβ antibody is complexed with an anti-blood brain barrier receptor/hapten bispecific antibody.

One aspect as reported herein is a non-covalent complex as reported herein for use as a medicament.

One aspect as reported herein is a non-covalent complex as reported herein for use in treating Alzheimer's disease.

One aspect as reported herein is a non-covalent complex as reported herein for use in the treatment of prodromal Alzheimer's disease.

One aspect as reported herein is a non-covalent complex as reported herein for use in the treatment of mild Alzheimer's disease.

One aspect as reported herein is a non-covalent complex as reported herein for alleviating Tau (pS422) induced neurodegeneration.

One aspect as reported herein is for maintaining cognition and function as reported herein. Guided non-covalent complexes.

One aspect as reported herein is a non-covalent complex as reported herein for slowing the rate of cognitive and dysfunction.

One aspect as reported herein is a non-covalent complex as reported herein for use in the manufacture of a medicament.

In one embodiment, the medicament is for treating Alzheimer's disease.

In one embodiment, the medicament is for treating prodromal Alzheimer's disease.

In one embodiment, the medicament is for treating mild Alzheimer's disease.

In one embodiment, the agent is used to alleviate Tau (pS422)-induced neurodegeneration.

In one embodiment, the medicament is used to maintain cognition and function.

In one embodiment, the agent is used to slow the rate of cognitive and dysfunction.

One aspect as reported herein is a method of treating an individual having Alzheimer's disease comprising administering to the individual an effective amount of a non-covalent complex as reported herein.

One aspect as reported herein is a method of alleviating Tau (pS422)-induced neurodegeneration in an individual comprising administering to the individual an effective amount of a non-covalent complex as reported herein to reduce Tau (pS422) induction. The nerves are degraded.

One aspect as reported herein is a method of maintaining the cognition and function of an individual comprising administering to the individual an effective amount of a non-covalent complex as reported herein to maintain cognition and function.

One aspect as reported herein is a method of slowing the rate of cognitive and dysfunctional decline in an individual comprising administering to the individual an effective amount of a non-covalent complex as reported herein to slow the rate of cognitive and dysfunction.

One aspect as reported herein is the use of a non-covalent complex as reported herein for alleviating Tau (pS422)-induced neurodegeneration.

One aspect as reported herein is the use of a non-covalent complex as reported herein for maintaining cognition and function.

One aspect as reported herein is the use of non-covalent complexes as reported herein for slowing cognitive and dysfunctional rates.

Non-covalent complexes as reported herein can be used to treat Alzheimer's disease.

Progress in inhibiting/alleviating Alzheimer's disease and neuropathology can be achieved using non-covalent complexes as reported herein.

Non-covalent complexes as reported herein can be used to prevent the progression of Alzheimer's disease or even to delay the progression of Alzheimer's disease.

In one embodiment, the non-covalent complex as reported herein i) binds to Tau (pS422) on brain sections of Tau (pS422) transgenic mice and Alzheimer's disease patients; and / Or labeled Tau (pS422) in Tau (pS422) transgenic cells.

One aspect as reported herein is a non-covalent complex of the amino acid sequence SEQ ID NO: 03 that specifically binds to human Tau (pS422).

Non-covalent complexes as reported herein specifically bind to/recognize early and late disease-related forms of human Tau (pS422).

One aspect as reported herein is the use of a non-covalent complex as reported herein for preventing the spread of human Tau (pS422)-associated Alzheimer's disease.

One aspect as reported herein is the use of a non-covalent complex as reported herein for reducing lysosomal membrane collapse.

One aspect as reported herein is the use of non-covalent complexes as reported herein for stabilizing and/or disintegrating stable lysosomal membranes induced by human Tau (pS422).

One aspect as reported herein is the use of a non-covalent complex as reported herein for preventing progression of Alzheimer's disease.

Non-covalent complexes as reported herein function by antibody-mediated inhibition of human Tau (pS422) vaccination and spread between cells.

Non-covalent complexes as reported herein are protected by binding to human Tau (pS422) Protect the lysosome from fibril damage.

Figure 1: Sequence alignment of rabbit and humanized light chain variable domains; CDRs are framed.

Figure 2: (A) and (B) : Sequence alignment of rabbit and humanized heavy chain variable domains; CDRs are framed.

Figure 3: Biochemistry of different combinations of humanized VH and VL with (A) phosphorylated tau peptide, (B) phosphorylated full-length human tau, (C) unphosphorylated tau peptide, (D) unphosphorylated full-length human tau Binding; (1) = VH00 / VL00, (2) = VH32 / VL21, (3) = VH20 / VL22, (4) = VH32 / VL22, (5) = VH33 / VL22; coating concentration: phosphorylated tau peptide : 50 ng/ml, all other targets: 1 μg/ml; (If phosphorylated tau peptide was coated with 1 μg/ml, comparable results were obtained (data not shown)).

Figure 4: Different combinations of humanized VH and VL with (A) = full-length human tau S422A mutation, (B) = aggregated human Tau; (1) = VH00 / VL00, (2) = VH32 / VL21, (3) = VH20/VL22, (4) = VH32 / VL22, (5) = VH33 / VL22; coating concentration: phosphorylated tau peptide: 50 ng / ml, all other targets: 1 μg / ml; (if 1 μg / ml coated phosphoric acid The tau peptide was obtained with comparable results (data not shown)).

Figure 5: Western blot method shows the selectivity of the selected humanized VH/VL combination; (1) = VH00 / VL00, (2) = VH32 / VL21, (3) = VH20 / VL22, (4) = VH32 / VL22, (5) = VH33 / VL22.

Figure 6: Hyperphosphorylation of tau in brain extracts of patients with Alzheimer's disease; (1) = VH00 / VL00, (2) = VH32 / VL21, (3) = VH32 / VL22.

Figure 7: Flow of the blood brain barrier-shuttle module composition.

Figure 8: SEC characteristics and SDSPAGE of the blood brain barrier-swirl module as produced in Example 14.

Figure 9: Results of FACS analysis using hCMEC/D3 cells as TfR-expressing BBB source cell lines and Dig-Cy5 as a fluorescent payload.

Figure 10: Transcytosis of the hapten-binding bispecific antibody blood-brain barrier-shuttle module and release from endothelial cells; A: anti-CD33-dig antibody transwell assay, huFc ELISA; B: anti-TfR1 antibody swell assay, huFc ELISA; C: anti-TfR1 antibody-Dig swell assay, huFc ELISA; D: anti-TfR2 antibody swell assay, huFc ELISA; E: anti-TfR2 antibody Dig Transfusion analysis, huFc ELISA.

Figure 11: A: Composition and quantification of bispecific antibody-haptenized payload non-covalent complexes; B: Transcytosis of haptenized payloads using bispecific antibodies with reduced affinity for TfR and Release from endothelial cells (A: anti-CD33-Dig+Dig-DNA swell assay, qPCR; B: anti-CD33-Bio+Bio-DNA swell assay, qPCR, C: anti-TfR2-Dig+Dig - DNA swell assay, qPCR, D: anti-TfR2-Bio+Bio-DNA swell assay, qPCR).

Figure 12: Transcytosis and release from endothelial cells of a non-releasable blood-brain barrier-swing module with high affinity for TfR; A: anti-TrF1-Dig+Dig-DNA Weil assay, qPCR, B: anti-TfR1 antibody - Bio+Bio-DNA, assay, qPCR).

Figure 13: The haptenization payload from the binding, uptake and intracellular separation of the non-releasable blood-brain barrier-swirl module with high affinity for TfR; shown in hCMEC/D3 cells after three hours of incubation at 37 °C Subcellular separation of bispecific antibody-complexed haptenated fluorescent payload. DIG-DNA-CY5 or Bio-DNA-Cy5 (dark gray) appeared in different intracellular vesicles and did not overlap with internalized anti-digoxigenin- or avidin-binding bispecific antibody (medium gray).

Figure 14: SDS PAGE gel of antibody 0155 coupled with HeliCar motif amino acid sequence cysteine variant 2 using a 2.5 mol excess of a compound containing a HeliCar motif amino acid sequence to form a covalent complex 0156; =HeliCar motif amino acid sequence cysteine variant 2; 2 = antibody 0019; 3 = antibody 0155.

Figure 15: SDS PAGE gel of antibody 0157 coupled with HeliCar motif amino acid sequence cysteine variant 1; 1 = HeliCar motif amino acid sequence cysteine variant 1 (oxidation); 2= Control coupling (oxidation); 3 = covalent conjugate (oxidation); 4 = molecular weight marker; 5 = covalent conjugate (reduction); 6 = control coupling (reduction); 7 = HeliCar motif amino acid sequence half Cysteine variant 1 (reduction).

Figure 16: Antibody 0155, a C-terminal acyl acid residue having SEQ ID NO: 28 deleted from the Pseudomonas aeruginosa exotoxin molecule LR8M containing the HeliCar motif amino acid sequence cysteine variant 1 and covalent conjugate thereof SEC chromatogram.

Figure 17: Analysis of the binding efficiency of non-reduced samples by SDS-CE, Caliper.

Figure 18: A: SEC-MALLS analysis was performed to identify and characterize complexes of anti-TfR/BRDU bispecific antibodies with BRDU-labeled DNA as well as free bispecific antibodies and free BRDU-DNA. A complex of MW of 244.9 kDa was eluted from the column, a free bispecific antibody with a MW of 215.4 kDa was detected and free BRDU-DNA with a MW of 16.4 kDa was detected.

B: SEC-MALLS analysis was performed to identify and characterize complexes of anti-TfR/BRDU bispecific antibodies with BRDU-labeled DNA as well as free bispecific antibodies and free BRDU-DNA. The complex exhibited a hydrodynamic radius of 6.8 nm, while the free bispecific antibody exhibited a hydrodynamic radius of 6.2 nm.

Figure 19: A: SEC-MALLS analysis to identify and characterize the complex of anti-TfR/biotin bispecific antibody with biotinylated anti-pTau antibody and free bispecific antibody and free biotinylated anti-pTau antibody . The complex exhibited a hydrodynamic radius of 8.0 nm, while the free bispecific antibody exhibited a hydrodynamic radius of 6.2 nm and the free biotinylated anti-pTau antibody exhibited a hydrodynamic radius of 5.5 nm.

B: SEC-MALLS analysis was performed to identify and characterize the complex of the anti-TfR/biotin bispecific antibody with the biotinylated anti-pTau antibody as well as the free bispecific antibody and the free biotinylated anti-pTau antibody. Self-manifold separation of MW 501kDa composite, detection A free bispecific antibody with a MW of 205 kDa was detected and a free biotinylated anti-pTau antibody with a MW of 150 kDa was detected.

C: If a wrong combination of hapten and anti-hapten antibody is used, no complex is formed.

Figure 20: Complex of biotinylated anti-pTau antibody with anti-CD33/biotin bispecific antibody (top left panel) and free biotinylated anti-pTau antibody (top right panel) without effective endocytosis (cytolysis, line) It was not transferred to the bottom outer side (left column, light gray) or the top (right column, black) compartment (load 3.8 μg/ml).

Complexation of biotinylated anti-pTau antibody with anti-TfR/biotin bispecific antibody 1 (bottom left panel) or anti-TfR/biotin bispecific antibody 2 (bottom right panel) mediated efficient endocytosis (cytolysis, Line) and then the biotinylated anti-pTau antibody was delivered to the bottom outer side (left column, light gray) and back to the top (right column, black) compartment (load 3.8 μg/ml).

Figure 21: Results of transcytosis assay of bispecific anti-human Tau (pS422)/biotin antibody non-covalent complexes and biotinylated anti-TfR antibody Fab fragments.

I. Definition

As used herein, the heavy and light chains are all according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acid positions of the constant regions and domains are numbered and referred to herein as "based on Kabat numbering." Specifically, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) Kabat numbering system (see pages 647-660) for kappa and The lambda isoform light chain constant domain CL and the Kabat EU index numbering system (see pages 661-723) are used for the constant heavy chain domains (CH1, hinge, CH2 and CH3).

For the purposes of this document, a "receptor human framework" is a light chain variable domain (VL) framework or heavy chain variable domain (VH) comprising a human immunoglobulin framework or a human consensus framework (as defined below). The framework of the framework amino acid sequence. The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence of a human immunoglobulin framework or a human consensus framework, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework sequence is identical in sequence to the VL human immunoglobulin framework sequence or the human co-framework.

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between a binding pair (eg, an antibody and an antigen) member, unless otherwise indicated. The affinity of molecule X for its conjugate Y is generally represented by the dissociation constant (kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity mature" anti-system refers to one or more of one or more hypervariable regions (HVRs) compared to one or more parent antibodies that do not have one or more changes in one or more hypervariable regions (HVRs) A variant of the antibody that alters the affinity of the antibody for the antigen.

The terms "anti-human Tau (pS422) antibody" and "antibody that specifically binds to human Tau (pS422)" refer to the ability to bind human Tau (pS422) with sufficient affinity to make the antibody suitable for targeting human Tau (pS422). And/or an antibody to a therapeutic agent. In one embodiment, the anti-human Tau (pS422) antibody binds to an unrelated non-human Tau (pS422) protein to a lesser extent than the antibody to human Tau (pS422), as measured, for example, by radioimmunoassay (RIA). About 10% of the degree.

The term "antibody" is used herein in its broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, as long as they exhibit The desired antigen binding activity can be.

"Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; bifunctional antibodies; linear antibodies; single-chain antibody molecules (eg, scFv); Multispecific antibodies.

The term "biotin", abbreviated as "BI", means 5-[(3aS,4S,6aR)-2-yloxyhexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid . Biotin is also known as vitamin H or coenzyme R.

The term "biotinylated antibody that specifically binds to human Tau (pS422)" means a binding entity comprising a biotin moiety, a linking group optionally present, and an antibody that specifically binds to human Tau (pS422). The linking group can be any linking group such as a peptide linking group or a chemical linking group.

The term "bispecific antibody" means an antibody having two different (antigen/hapten) binding specificities. In one embodiment, the bispecific antibody is specific for two different antigens (ie, haptens and non-hapten antigens).

The term "bromodeoxyuridine", abbreviated as "BrdU", means 5-bromo-2'-deoxyuridine. Bromo-deoxyuridine is also known as bromouridine, BudR, BrdUrd.

The term "bromo-deoxyuridine antibody specifically binding to human Tau (pS422)" means a combination comprising a bromodeoxyuridine moiety, a linking group optionally present, and an antibody that specifically binds to human Tau (pS422). entity. The linking group can be any linking group such as a peptide linking group or a chemical linking group.

The term "digoxigenin", abbreviated as "DIG", means 3-[(3S,5R,8R,9S,10S,12R,13S,14S,17R)-3,12,14-trihydroxy-10,13- Dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetrahydro-cyclopenta[a]-phenanthrene-17-yl]- 2H-furan-5-one (CAS No. 1672-46-4). Digoxin (DIG) is a steroid found in the flowers and leaves of the plants Digitalis purpurea, Digitalis orientalis, and Digitalis lanata (foxgloves). (Polya, G., Biochemical targets of plant bioactive compounds, CRC Press, New York (2003) p. 847).

The term "digoxigenin antibody that specifically binds to human Tau (pS422)" means a digoxin moiety, a linking group optionally present, and a specific binding to human Tau. The binding entity of the antibody (pS422). The linking group can be any linking group such as a peptide linking group or a chemical linking group.

The term "luciferin", abbreviated as "FLUO", means 6-hydroxy-9-(2-carboxyphenyl)-(3H)-dibenzopyran-3-one, or 2-(6-hydroxy- 3-Sideoxy-(3H)-dibenzopyran-9-yl)-benzoic acid. Luciferin is also known as Fluorescent Yellow, C.I.45350, Solvent Yellow 94, D & C No. 7 Yellow, Angiophor, Japan Yellow 201 or Soap Yellow.

The term "luciferylated antibody that specifically binds to human Tau (pS422)" denotes a binding entity comprising a luciferin moiety, a linking group optionally present, and an antibody that specifically binds to human Tau (pS422). The linking group can be any linking group such as a peptide linking group or a chemical linking group.

The term "theophylline", abbreviated as "THEO", means 1,3-dimethyl-7H-indole-2,6-dione. Theophylline is also known as dimethylxanthine.

The term "theoylated antibody that specifically binds to human Tau (pS422)" denotes a binding entity comprising a theophylline moiety, optionally a linking group, and an antibody that specifically binds to human Tau (pS422). The linking group can be any linking group such as a peptide linking group or a chemical linking group.

The term "hapten" means a small molecule that elicits an immune response only when attached to a large carrier such as a protein. Exemplary haptens are aniline, o-, m- and p-aminobenzoic acid, hydrazine, histamine-butadienyl-glycine (HSG), pyridazine, halothane, indium-DTPA, luciferin, organism , digoxin, theophylline, bromodeoxyuridine and dinitrophenol. In one embodiment, the hapten is biotin or digoxin or theophylline or luciferin or bromodeoxyuridine.

The term "haptenized antibody that specifically binds to human Tau (pS422)" means (covalently) a hapten that binds to an antibody that specifically binds to human Tau (pS422). Activated hapten derivatives are useful as starting materials for the formation of such conjugates. In one embodiment, the hapten is bound via a linking group (in one embodiment via its 3-hydroxyl group) to an antibody that specifically binds to human Tau (pS422). In one embodiment, the linking group comprises a) one or more (in In one embodiment three to six) methylene-carboxy-methyl (-CH2-C(O)-), and/or b) 1 to 10 (in one embodiment 1 to 5) amino acids a residue (in one embodiment selected from the group consisting of glycine, serine, glutamic acid, beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid or lysine), and/or c) one or more (in one embodiment, one or two) compounds having the formula NH2-[(CH2)nO]xCH2-CH2-COOH, wherein n is 2 or 3 and x is from 1 to 10, in In one embodiment, 1 to 7. The last element results in (at least in part) a linking group (partially) of the formula -NH-[(CH2)nO]xCH2-CH2-C(O)-. An example of such a compound is, for example, 12-amino-4,7,10-trioxadecanoic acid (resulting in a TEG (triethylene glycol) linking group). In one embodiment, the linking group additionally comprises a maleimide group. In addition, the linking group can spatially aid in the binding of the anti-hapten antibody to the hapten that specifically binds to the haptenated antibody of human Tau (pS422). In one embodiment, the linking group binds to an amino acid side chain of an antibody that specifically binds to human Tau (pS422) (eg, bound to an amino acid or cysteine side chain via an amine or thiol group) . In one embodiment, the linking group binds to the amine or carboxy terminus of an antibody that specifically binds to human Tau (pS422). The binding site of the linking group to an antibody that specifically binds to human Tau (pS422) is typically selected in a region that binds to the linking group and does not affect the biological activity of the antibody that specifically binds to human Tau (pS422). Thus, the position of attachment of the linking group will depend on the relevant structural element responsible for the biological activity of the antibody that specifically binds to human Tau (pS422). The biological activity of an antibody that specifically binds to human Tau (pS422) linked to a hapten can be tested in an in vitro assay before or after binding.

The term "chimeric" anti-system refers to a portion of the heavy chain and/or light chain that is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from antibodies of different sources or species.

The "class" of an antibody refers to the type of constant or constant region that its heavy chain has. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

"Effective function" refers to the biological activity attributable to the Fc region of an antibody as a function of antibody class. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors) ) down-regulation; and B cell activation.

An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve the desired treatment or control result at the necessary dosage and for a sustained period of time.

The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminus of the Fc region may or may not be present in the amine acid (Lys447). Unless otherwise stated herein, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, also known as the EU index, as in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), described in NIH Publication 91-3242.

"Framework" or "FR" refers to a variable domain residue other than a hypervariable region (HVR) residue. The FR of the variable domain is usually composed of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full length antibody", "intact antibody" and "complete antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein. The term "full length antibody" denotes a polypeptide consisting of two antibody light chain polypeptides joined by a disulfide bond and two antibody heavy chain polypeptides, wherein the C-terminal amino acid residues are present in the two antibody heavy chain polypeptides ( K) may or may not be present.

The terms "host cell," "host cell strain," and "host cell culture" are used interchangeably herein and refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and their derived progeny regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as for screening or selection in the original transformed cell.

The "human common framework" is a framework representing the most frequently occurring amino acid residues in the selected human immunoglobulin VL or VH framework sequences. In general, the human immunoglobulin VL or VH sequence is selected from a subpopulation of variable domain sequences. In general, the subgroup of sequences is a subgroup of, for example, Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed., Bethesda MD (1991), NIH Publication 91-3242, Volumes 1-3. In one embodiment, for VL, the subgroup is a subgroup κI as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. (supra).

A "humanized" anti-system refers to a chimeric antibody comprising an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (eg, CDRs) correspond to HVRs of non-human antibodies, and all or substantially All FRs above correspond to the FR of human antibodies. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

The term "hypervariable region" or "HVR" as used herein refers to a sequence that is hypervariable in the variable domain of an antibody ("complementarity determining region" or "CDR") and forms a structurally defined loop ("hypervariable loop"), And/or regions containing antigen-contacting residues ("antigen contacts"). In general, antibodies contain six HVRs; three are located in VH (H1, H2, H3), and three are located in VL (L1, L2, L3).

The HVR herein includes (a) occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and Hypervariable loop at 96-101 (H3) (Chothia, C. and Lesk, AM, J. Mol. Biol. 196 (1987) 901-917); (b) appears at amino acid residues 24-34 ( CDRs at L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, EA et al., Sequences of Proteins Of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242); (c) appears in amino acid residues 27c-36 (L1), 46-55 Antigen contacts at (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732- 745 (1996)); and (d) a combination of (a), (b) and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains herein are numbered according to the above-referenced literature by Kabat et al.

An "immunoconjugate" is an antibody that binds to one or more heterologous molecules.

"Individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates, such as monkeys), rabbits, and rodents (eg, small Rat and rat). In certain embodiments, the individual is a human.

An "isolated" antibody is an antibody that is separated from components of the natural environment. In some embodiments, the anti-system is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC). Measured. For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87. "Separated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-human Tau (pS422) antibody" refers to one or more nucleic acid molecules encoding an antibody heavy and light chain (or a fragment thereof), including such nucleic acid molecules in a single vector or in separate vectors. And such nucleic acid molecules present at one or more locations in the host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, a variant variant antibody (eg, containing a naturally occurring mutation or a variant occurring during the production of a monoclonal antibody preparation). In addition to antibodies, such variants are typically present in minor amounts, the individual antibodies comprising the population are identical and/or bind to the same epitope. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody against the individual antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "single plant" indicates the property of the anti-system obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, fusion knob methods, recombinant DNA methods, phage display methods, and utilization of all or part of human immunoglobulins. Methods of transgenic animal loci, such methods and other exemplary methods of making monoclonal antibodies are described herein.

"Native antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains that are disulfide-bonded and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. antibody Light chains can be classified into one of two types, called kappa and lambda, based on their amino acid sequence in the constant domain.

The term "pharmaceutical instructions" is used to mean a specification that is typically included in a commercial package of a therapeutic product, which contains indications, usage, dosage, dosing, combination therapy, contraindications and/or related to the use of such therapeutic products. Warning information.

"Percent amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as after aligning the reference polypeptide sequence with the candidate sequence and, if necessary, introducing a gap to achieve a maximum sequence identity percentage, and without any conservation The percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence in the case of substitution as part of sequence identity. The alignment can be accomplished in a variety of ways within the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software to determine the percent identity of the amino acid sequence. Those skilled in the art can determine parameters suitable for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this document, the sequence comparison computer program ALIGN-2 was used to generate amino acid sequence identity percent values. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with the User's Guide at U.S. Copyright Office (Washington D.C., 20559), which is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are unchanged.

In the case of amino acid sequence comparison using ALIGN-2, the amino acid sequence identity % of the given amino acid sequence A for, or with respect to the predetermined amino acid sequence B is calculated as follows (or may be referred to as established The amino acid sequence A has or contains a certain amino acid sequence identity for,, or relative to, a predetermined amino acid sequence B: 100 fractions X/Y

Wherein X is the number of amino acid residues rated as uniformly matched when the sequence alignment program ALIGN-2 is used to align A and B, and wherein Y is the total number of amino acid residues in B. It will be appreciated that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the % identity of the amino acid sequence of A and B will not be equal to the % identity of the amino acid sequence of B and A. All amino acid sequence identity % values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless otherwise specifically stated.

The term "pharmaceutical formulation" refers to a formulation that is in a form that allows for the biological activity of the active ingredient contained therein to be effective and free of other components that are unacceptably toxic to the individual to which the formulation is administered.

"Pharmaceutically acceptable carrier" means a component of a pharmaceutical formulation that is not toxic to an individual other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "human Tau (pS422)" as used herein refers to native human Tau (pS422) (UniProt P37840). The term encompasses "full length" untreated human Tau (pS422) as well as any form of human Tau (pS422) produced by treatment in cells. The term also encompasses variants of naturally occurring human Tau (pS422), such as mutations, splice variants or dual gene variants. The amino acid sequence of human Tau (pS422) is shown in SEQ ID NO:02.

As used herein, "treatment" (and grammatical variants thereof, such as "treat" or "treating" refers to clinical interventions that attempt to alter the natural course of the individual being treated, and may be performed For prevention or in the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating the symptoms, alleviating any direct or indirect pathological results of the disease, preventing metastasis, reducing the rate of disease progression, Improving or alleviating disease conditions, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay disease production or slow disease progression.

The term "x price", such as "unit price" or "two price" or "three price" or "four price", Indicates that a specified number of binding sites are present in the antibody molecule, ie, "x". Thus, the terms "bivalent", "tetravalent" and "hexavalent" mean that there are two binding sites, four binding sites and six binding sites in the antibody molecule, respectively. Bispecific antibodies are at least "bivalent" and can be "trivalent" or "multivalent" (eg "tetravalent" or "hexavalent"). In one embodiment, the bispecific antibody is bivalent, trivalent or tetravalent. In one embodiment, the bispecific antibody is bivalent. In one embodiment, the bispecific antibody is trivalent. In one embodiment, the bispecific antibody is tetravalent.

Bispecific antibodies can be bispecific even in the presence of more than two binding sites (ie, antibodies are trivalent or multivalent). The term bispecific antibodies include, for example, multivalent single chain antibodies, bifunctional antibodies and trifunctional antibodies, as well as antibodies having a constant domain structure of full length antibodies linked to other antigen binding sites via one or more peptide linking groups. (eg, single-chain Fv, VH domain and/or VL domain, Fab or (Fab) 2). The antibody can be a full length antibody from a single species, or a chimeric or humanized antibody. For antibodies with more than two antigen binding sites, some of the binding sites may be the same as long as the antibody has binding sites for two different antigens. That is, since the first binding site is specific for the hapten, the second binding site is specific for the non-hapten antigen and vice versa.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of the native antibody (VH and VL, respectively) generally have a similar structure, wherein each domain comprises four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt, T. J. et al., Kuby Immunology, 6th ed., W. H. Freeman and Co., N. Y. (2007), p. 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using VH or VL domains from antibodies that bind antigen to separately screen libraries of complementary VL or VH domains. See, for example, Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

The term "vector," as used herein, refers to a nucleic acid molecule that is capable of transmitting Another nucleic acid to which it is attached. The term includes a vector in the form of a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it is introduced. Certain vectors are capable of directing the performance of the nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression carriers."

II. Compositions and methods A. Blood-brain barrier shuttle as reported herein

A portion of the non-covalent complex as reported herein is the blood brain barrier-shuttle module (BBB-shuttle module), which has a first binding specificity for the hapten and a blood brain barrier receptor ( A second binding specificity bispecific antibody of BBBR). Such BBB-shuttle modules recognize cell surface targets (such as TfR, LRP or other targets, BBBR) that are capable of transcytosis on the blood-brain barrier and simultaneously bind to the haptenation payload.

No more requirements regarding binding valence, antibody type, BBBR binding affinity have been found to be met.

It has also been discovered that a bispecific antibody-based shuttle module as reported herein does not have to be released from endothelial cells of the blood-brain barrier to mediate transcytosis of the hapten-loaded payload. In fact, the haptenation payload of the shuttle module based on the bispecific antibody complex/binding to the bispecific antibody-based shuttle module is based on the bispecific antibody from the BBB cell when bound to the BBBR. The shuttle module is released, i.e., in the intracellular vesicle system, separated from the shuttle module, and subsequently excreted from the BBB cells into the brain leaving bispecific antibodies in the BBB cells.

The bispecific antibody-based shuttle module as reported herein is highly variable in terms of BBBR binding specific valency and BBBR binding specific affinity. At the same time, it enables the payload to be released from the shuttle module.

Multispecific antibody

A variety of recombinant antibody formats (eg, tetravalent bispecific antibodies) have been developed by fusing, for example, IgG antibody versions with single-stranded domains (see, for example, Coloma, M. J. et al., Nature). Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S. L., Nature Biotech 25 (2007) 1233-1234).

In addition, several other versions have been developed that no longer retain the core structure of the antibody (IgA, IgD, IgE, IgG or IgM), such as difunctional, trifunctional or tetrafunctional antibodies, minibodies, several single-stranded forms (scFv, Bis-scFv), which is capable of binding two or more antigens (Holliger, P. et al, Nature Biotech 23 (2005) 1126-1136; Fischer, N., Léger, O., Pathobiology 74 (2007) 3- 14; Shen, J., et al, Journal of Immunological Methods 318 (2007) 65-74; Wu, C. et al, Nature Biotech. 25 (2007) 1290-1297).

All of this type uses a linker fusion antibody core (IgA, IgD, IgE, IgG or IgM) with another binding protein (eg scFv) or a fusion such as two Fab fragments or scFv (Fischer, N. and Léger, O. , Pathobiology 74 (2007) 3-14) or CrossFab. It must be remembered that it may be desirable to retain effector functions mediated via Fc receptor binding, such as complement dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity, by maintaining a high degree of similarity to naturally occurring antibodies ( ADCC).

Dual variable domain immunoglobulins as engineered multivalent and multi-specific binding proteins are reported in WO 2007/024715. A method of preparing a bioactive antibody dimer is reported in US 6,897,044. Multivalent FV antibody constructs having at least four variable domains joined to each other by a peptide linking group are reported in US 7,129,330. Dimeric and multimeric antigen binding structures are reported in US 2005/0079170. No. 6,511,663 reports a trivalent or tetravalent monospecific antigen binding protein comprising three or four Fab fragments covalently linked to each other by a linker structure which is not a native immunoglobulin. Tetravalent bispecific antibodies that are effective in prokaryotic and eukaryotic cells and are suitable for use in therapeutic and diagnostic methods are reported in WO 2006/020258. US 2005/0163782 reports a dimerization from a mixture comprising two polypeptide dimers from a dimer that is not separated by at least one interchain disulfide linkage or preferably synthesized via at least one interchain disulfide linkage. The method of body. Bispecific tetravalent receptors are reported in US 5,959,083. Engineered antibodies with three or more functional antigen binding sites are reported in WO 2001/077342.

Multispecific and multivalent antigen binding polypeptides are reported in WO 1997/001580. WO 1992/004053 reports that homoconjugates prepared from IgG-based monoclonal antibodies that bind to the same antigenic determinant are covalently linked by synthetic cross-linking. An oligomeric monoclonal antibody having high affinity for an antigen is reported in WO 1991/06305, whereby an IgG-like oligomer is secreted, which has two or more immunoglobulin monomers associated with each other to form four A valence or hexavalent IgG molecule. Sheep-derived antibodies and engineered antibody constructs are reported in US 6,350,860, which are useful in the treatment of pathogenic diseases of interferon gamma activity. A targetable construct as a multivalent vector for a dual specific antibody is reported in US 2005/0100543, and each molecule of the targeted construct can serve as a vector for two or more dual specific antibodies. . A genetically engineered bispecific tetravalent antibody is reported in WO 1995/009917. Stable binding molecules consisting of or comprising a stable scFv are reported in WO 2007/109254.

In certain embodiments, an antibody provided herein or an antibody in a conjugate as reported herein is a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for a hapten and the other is for any other (non-hapten) antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for the preparation of multispecific antibodies include, but are not limited to, recombinantly co-presenting two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983). 537-540; WO 93/08829; and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "杵-臼" engineering (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by engineering engineered electrostatic steering effects of antibody Fc-heterodimeric molecules (WO 2009/089004); crosslinking two or more antibodies or fragments (see, for example, US 4,676,980 and Brennan, M. et al, Science 229 (1985) 81-83); production of bispecific antibodies using leucine zippers (see, eg, Kostelny, SA et al, J. Immunol. 148 (1992) 1547-1553); preparation of bispecific antibody fragments "Bifunctional antibody" technology (see, eg, Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and the use of single-chain Fv (scFv) dimers (see, eg, Gruber, M et al, J. Immunol. 152 (1994) 5368-5374); and preparation of trispecific antibodies as described, for example, in Tutt, A. et al. J. Immunol. 147 (1991) 60-69.

In one embodiment, the CH3 domain of the heavy chain of the bispecific antibody is altered by the "杵-臼" technique, for example, in WO 96/027011, WO 98/050431, Ridgway JB, et al., Protein Eng. 9 (1996) 617-621, Merchant, AM et al, Nat Biotechnol 16 (1998) 677-681 are described in detail by several examples. In this method, the interaction surfaces of the two CH3 domains are altered to increase heterodimerization of the two heavy chains containing the two CH3 domains. Each of the two CH3 domains (of the two heavy chains) may be "杵" and the other may be "臼". Introduction of disulfide bridges stabilizes heterodimers (Merchant, AM et al, Nature Biotech 16 (1998) 677-681; Atwell, S. et al, J. Mol. Biol. 270 (1997) 26-35) and Increase production.

In one embodiment of all aspects, the bispecific antibody is characterized in that - the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each meet at an interface comprising an initial interface between the CH3 domains of the antibody, Wherein the interface is altered to facilitate the formation of a bispecific antibody, wherein the alteration is characterized by a) a change in the CH3 domain of a heavy chain such that within the initial interface, the CH3 domain of one heavy chain encounters another heavy in the bispecific antibody At the initial interface of the CH3 domain of the chain, an amino acid residue is replaced by an amino acid residue having a larger side chain volume to create a protuberance in the interface of the CH3 domain of a heavy chain. Convex can be located a recess in the interface of the CH3 domain of the other heavy chain; and b) a change in the CH3 domain of the other heavy chain such that the second CH3 structure of the initial interface of the first CH3 domain is encountered within the bispecific antibody Within the initial interface of the domain

An amino acid residue is displaced by an amino acid residue having a smaller side chain volume to create a pocket in the interface of the second CH3 domain, and a crown in the interface of the first CH3 domain can Located in the pocket.

Thus, in one embodiment, an antibody as reported herein is characterized by - the initial of the CH3 domain of the first heavy chain of the full length antibody and the CH3 domain of the second heavy chain of the full length antibody, respectively, between the antibody CH3 domains The interface contains a changed interface, where i) is in the CH3 domain of the first heavy chain

An amino acid residue is displaced by an amino acid residue having a larger side chain volume to create a protuberance in the interface of the CH3 domain of one heavy chain, which may be located in the CH3 structure of the other heavy chain In a pocket within the interface of the domain; and wherein ii) is in the CH3 domain of the second heavy chain

An amino acid residue is displaced by an amino acid residue having a smaller side chain volume to create a pocket in the interface of the second CH3 domain, and a crown in the interface of the first CH3 domain can Located in the pocket.

In one embodiment, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan ( W).

In one embodiment, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), proline ( V).

In one embodiment, the two CH3 domains are further altered by introducing cysteine (C) as the amino acid in the corresponding position of each CH3 domain, such that it can be in two CH3 A disulfide bridge is formed between the domains.

In a preferred embodiment, the multispecific antibody comprises an amino acid T366W mutation in the first CH3 domain of the "purine chain" and an amino acid T366S, L368A in the second CH3 domain of the "purine chain", Y407V mutation. For example, by introducing an amino acid Y349C mutation into the CH3 domain of the "臼 chain" and introducing an amino acid E356C mutation or an amino acid S354C mutation into the CH3 domain of the "杵 chain", the CH3 domain can also be used. Additional interchain disulfide bridges (Merchant, AM et al, Nature Biotech. 16 (1998) 677-681).

In one embodiment, the bispecific antibody comprises the Y349C, T366W mutation in one of the two CH3 domains and the E356C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In one embodiment, the bispecific antibody comprises the Y349C, T366W mutation in one of the two CH3 domains and the S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains (one CH3) An additional Y349C mutation in the domain forms an interchain disulfide bridge with an additional E356C or S354C mutation in another CH3 domain) (according to the EU index of Kabat; Kabat, EA et al, Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Other 杵-臼 techniques as described in EP 1 870 459 A1 may alternatively or additionally be used. Thus, another example of a bispecific antibody is the R409D, K370E mutation of the CH3 domain of the "杵 chain" and the D399K, E357K mutation in the CH3 domain of the "臼 chain" (according to the EU index number of Kabat; (Kabat, EA et al, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

In one embodiment, the bispecific antibody comprises a T366W mutation in the CH3 domain of the "杵 chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "臼 chain", and a CH3 domain of the "杵 chain" Additional R409D, K370E mutations and D399K, E357K mutations in the CH3 domain of the "臼 chain".

In one embodiment, the bispecific antibody comprises a Y349C, T366W mutation in one of two CH3 domains and a S354C, T366S, L368A, Y407V mutation or bispecific in the other of the two CH3 domains The antibody comprises the Y349C, T366W mutation in one of the two CH3 domains and the S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains and the CH3 domain of the "杵 chain" Additional R409D, K370E mutations and D399K, E357K mutations in the CH3 domain of the "臼 chain". Such 杵 and 臼 mutations in the CH3 domain are commonly used in the human heavy chain constant region of SEQ ID NO: 58 (human IgG1 subtype) (Caucasian and African-American (Afro-American) or mutant L234A /L235A, and L234A/L235A/P329G) (in accordance with Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) EU index number).

In one embodiment, the bispecific antibody comprises additional such "杵" and "臼" mutations in the CH3 domain (eg, Y349C, T366W mutations and two CH3 structures in one of the two CH3 domains) Human heavy chain constant region of SEQ ID NO: 58 of the S354C, T366S, L368A, Y407V mutation in the other of the domains (according to Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) EU index number).

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, for example, US 2006/0025576).

An antibody or fragment herein also includes a "double-affected Fab" or "DAF" comprising an antigen binding site that binds to a hapten and a different antigen (see, for example, US 2008/0069820).

The antibodies or fragments herein also include the multispecific antibodies described in: WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792 and WO 2010/145793.

In a preferred embodiment, the multispecific antibody (which comprises a CH3 domain in each heavy chain) comprises an amino acid S354C, a T366W mutation and two CH3 domains in one of the two CH3 domains The amino acid Y349C, T366S, L368A, Y407V mutations in the other (the additional amino acid S354C mutation in one CH3 domain and the additional amino acid Y349C mutation in another CH3 domain form an interchain disulfide bridge) ) (according to Kabat numbering).

Other techniques encompassing CH3 modification of heterodimerization are contemplated as alternatives and are described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304 WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment, the heterodimerization process described in EP 1 870 459 A1 is used. This method is based on the introduction of a substitution/mutation of an oppositely charged charged amino acid at a specific amino acid position in the CH3/CH3 domain interface between the two heavy chains. In a preferred embodiment, the multispecific antibody comprises an amino acid R409D, a K370E mutation in the CH3 domain of the first heavy chain (of the multispecific antibody) and a second heavy chain (of the multispecific antibody) Amino acid D399K, E357K mutations in the second CH3 domain (according to Kabat numbering).

In another embodiment, the multispecific antibody comprises the amino acid T366W mutation in the CH3 domain of the "purine chain" and the amino acid T366S, L368A, Y407V mutation in the CH3 domain of the "purine chain" and the other " The amino acid R409D, K370E mutation in the CH3 domain of the 杵 chain and the amino acid D399K and E357K mutations in the CH3 domain of the "臼 chain".

In another embodiment, the multispecific antibody comprises the amino acid S354C, T366W mutation in one of the two CH3 domains and the amino acid Y349C, T366S, L368A in the other of the two CH3 domains , Y407V mutant or multispecific antibody contains two CH3 junctions The amino acid Y349C, T366W mutation in one of the domains and the amino acid S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains and the CH3 domain of the other "杵 chain" Amino acid R409D, K370E mutation and amino acid D399K, E357K mutation in the CH3 domain of "臼 chain".

In one embodiment, the heterodimerization process described in WO 2013/157953 is used. In one embodiment, the first CH3 domain comprises an amino acid T366K mutation and the second CH3 domain comprises an amino acid L351D mutation. In another embodiment, the first CH3 domain additionally comprises an amino acid L351K mutation. In another embodiment, the second CH3 domain additionally comprises an amino acid mutation selected from the group consisting of Y349E, Y349D, and L368E (preferably L368E).

In one embodiment, the heterodimerization process described in WO2012/058768 is used. In one embodiment, the first CH3 domain comprises an amino acid L351Y, Y407A mutation and the second CH3 domain comprises an amino acid T366A, K409F mutation. In another embodiment, the second CH3 domain comprises another amino acid mutation at: T411, D399, S400, F405, N390 or K392, for example selected from the group consisting of a) T411N, T411R, T411Q, T411K, T411D , T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R or S400K, F405I, F405M, F405T, F405S, F405V or F405W, N390R, N390K or N390D, K392V, K392M, K392R, K392L , K392F or K392E. In another embodiment, the first CH3 domain comprises an amino acid L351Y, Y407A mutation and the second CH3 domain comprises an amino acid T366V, K409F mutation. In another embodiment, the first CH3 domain comprises an amino acid Y407A mutation and the second CH3 domain comprises an amino acid T366A, K409F mutation. In another embodiment, the second CH3 domain additionally comprises amino acid K392E, T411E, D399R and S400R mutations.

In one embodiment, the heterodimerization process described in WO2011/143545 is used with, for example, an amino acid modification selected from the group consisting of 368 and 409.

In one embodiment, the heterodimerization method described in WO2011/090762 is used, The 杵-臼 technique described above is also used. In one embodiment, the first CH3 domain comprises an amino acid T366W mutation and the second CH3 domain comprises an amino acid Y407A mutation. In one embodiment, the first CH3 domain comprises an amino acid T366Y mutation and the second CH3 domain comprises an amino acid Y407T mutation.

In one embodiment, the multispecific antibody has an IgG2 isotype and uses the heterodimerization method described in WO2010/129304.

In one embodiment, the heterodimerization process described in WO 2009/089004 is used. In one embodiment, the first CH3 domain comprises an amino acid residue K392 or N392 substituted with a negatively charged amino acid such as glutamic acid (E) or aspartic acid (D), preferably K392D or N392D And the second CH3 domain comprises an amino acid residue D399, E356, D356 or E357 via a positively charged amino acid (eg, lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K and better D399K and E356K) replaced. In another embodiment, the first CH3 domain additionally comprises an amino acid residue K409 or R409 via a negatively charged amino acid (eg, glutamic acid (E) or aspartic acid (D), preferably K409D or R409D ) replaced. In another embodiment, the first CH3 domain additionally or alternatively comprises an amino acid residue K439 and/or K370 substituted with a negatively charged amino acid such as glutamic acid (E) or aspartic acid (D) .

In one embodiment, the heterodimerization process described in WO2007/147901 is used. In one embodiment, the first CH3 domain comprises amino acid K253E, D282K and K322D mutations and the second CH3 domain comprises amino acid D239K, E240K and K292D mutations.

In one embodiment, the heterodimerization process described in WO2007/110205 is used.

B. Non-covalent complexes as reported herein

The blood-brain barrier shuttle as reported herein is used as an antibody that specifically binds to the human Tau (pS422) delivery vector. An antibody that specifically binds to human Tau (pS422) binds to a hapten and thus recombines via the hapten binding site of the blood brain barrier shuttle. This complex is defined and the haptenated antibody that specifically binds to human Tau (pS422) is stable and specific for transmission across the blood-brain barrier. Because of the specific binding to human Tau (pS422) haptenation The antibody complexes with the blood-brain barrier shuttle in a non-covalent manner. On the one hand, a haptenated antibody that specifically binds to human Tau (pS422) binds to its delivery medium during its cycle time (= blood-brain barrier shuttle = double Specific antibodies), but on the other hand can also be effectively released after transcytosis. Binding to a hapten can be achieved without interference with the activity of an antibody that specifically binds to human Tau (pS422). The blood-brain barrier shuttle does not contain abnormal covalent additions and thus avoids any immunogenic risk. A complex comprising a hapten-specific binding site specifically binding to human Tau (pS422) and a bispecific antibody as described herein confers benign antibodies that specifically bind to human Tau (pS422) Biophysical properties. In addition, such complexes are capable of targeting a load to a cell or tissue that exhibits an antigen specifically recognized by a second binding of a bispecific antibody.

Antibodies that specifically bind to human Tau (pS422) retain their functionality despite haptenation and despite the transmembrane shuttle complex (=bispecific antibody) via the blood brain barrier. Furthermore, the blood-brain barrier receptor binding site of the bispecific antibody retains its binding specificity and affinity in the presence of a complex haptenized antibody that specifically binds to human Tau (pS422). A complex of a haptenated antibody and a bispecific antibody that specifically binds to human Tau (pS422) as reported herein can be used to specifically target an antibody that specifically binds to human Tau (pS422) to express a blood-brain barrier. The body of the body. Since a haptenated antibody that specifically binds to human Tau (pS422) is coupled to a bispecific antibody in a non-covalent manner, an antibody that specifically binds to human Tau (pS422) can be released after internalization or transcytosis.

Subsequent processing of antibodies is very complex due to their chemical and physical properties, such as molecular weight and domain architecture (including secondary modifications). For example, not only do the formulated drugs but also the intermediates in the subsequent processing (DSP) concentrated solution need to achieve a low volume for economical disposal and application storage.

As the antibody concentration increases, the tendency to form aggregates can be observed. Such aggregated antibodies are characterized by attenuated characteristics compared to isolated antibodies. Aggregation of the complexes as reported herein can be introduced between the heavy and light chain variable domains of single-chain antibodies in the blood-brain barrier shuttle module Disulfide bonds are reduced. The stability of this improvement applies not only to the production process but also to the storage of the composite. In one embodiment, the disulfide bond between the variable domains of the single chain antibody comprised in the bispecific antibody is selected independently of: i) the heavy chain variable domain position 44 to the light chain variable domain. Position 100, ii) heavy chain variable domain position 105 to light chain variable domain position 43, or heavy chain variable domain position 101 to light chain variable domain position 100.

In one embodiment, the disulfide bond between the variable domains of the single chain antibody contained in the bispecific antibody is between the heavy chain variable domain position 44 and the light chain variable domain position 100.

In one embodiment, the disulfide bond between the variable domains of the single chain antibody contained in the bispecific antibody is between the heavy chain variable domain position 105 and the light chain variable domain position 43.

C. Exemplary antibodies that specifically bind to human Tau (PS422)

Humanized antibodies that specifically bind to human Tau (pS422) as non-covalent complexes as reported herein are not available by standard humanization methods. It is desirable to introduce a non-standard mutation into the amino acid sequence to obtain a humanized antibody having comparable binding characteristics to the parent rabbit antibody. This is especially important because antibodies as reported herein are intended to cross the human blood brain barrier and function in the human brain. Therefore, the general application criteria for selecting humanized antibodies are not so strict that they cannot be directly applied to the current situation.

It has been found that in order to obtain a suitable and developable humanized antibody, two cysteine acids which form a disulfide bridge in CDRL3 (light chain CDR3) must be replaced by serine and isoleucine, respectively. In addition to ensuring the proper orientation of the same CDRL3, the isoalanine residues present in the middle of the rabbit CDRL3 are also deleted, resulting in a humanized CDRL3 with one amino acid residue less than the parental rabbit CDRL3.

It has also been found to be advantageous to maintain three glutamic acid amino acid residues in the heavy chain at positions 4, 24 and 78. Without being bound by this theory, it is assumed that such residues are required to ensure proper expression of the antigen binding loop of the heavy chain variable region. Additionally, the presence of arginine residues at position 71 is advantageous.

Sequence alignments of different humanized light chain variable domains are shown in Figure 1. Different human weight Sequence alignment of the chain variable domains is shown in Figure 2. All numbers as used herein are based on the Kabat variable domain numbering scheme.

In the table below, the characteristics of different humanized variants of the rabbit light chain variable domain in combination with the humanized heavy chain variable domains VH14 and VH20 are shown, respectively. The binding partner is human Tau (pS422).

Reference values VH00 and VL00 (rabbit antibody): 25 ° C: kd = 2.6 E-04; t / 2 = 44 min.

37 ° C: ka = 3.7E + 04, kd = 5.25E-03, KD = 1.4E-08, t / 2 = 22 min.

In the table below, the characteristics of different humanized variants of the domestic light-free chain variable domain in combination with the humanized light chain variable domains VL17 and VL19 are shown, respectively.

Reference values VH00 and VL00 (rabbit antibody): 25 ° C: kd = 2.6 E-04; t / 2 = 44 min.

37 ° C: ka = 3.7E + 04, kd = 5.25E-03, KD = 1.4E-08, t / 2 = 22 min.

In the table below, the kinetic constants for the different VH/VL combinations are shown.

The biochemical combination of different combinations of humanized VH and VL is shown in Figures 3 and 4.

The binding specificity of different VH/VL combinations (EC50 values in [ng/ml]) are shown in the table below.

The sensitivity of the selected humanized VH/VL combination to the human tau mutation S422A is visible from the western blot shown in Figure 5. All humanized variants selectively bind to human tau phosphorylated at S422. There is a low level of x reactivity to the non-S422 phosphorylation epitope of the parental rabbit antibody, while the humanized variants shown have a lower cross-reactivity in this respect than the parental rabbit antibody.

Show parental rabbit antibody and selected humanized anti-human Tau (pS422) antibody in Figure 6. Binding to PHF-tau in brain extracts of patients with Alzheimer's disease.

The table below summarizes the biological characteristics of the selected humanized VH/VL combination.

In one embodiment, an antibody that specifically binds to human Tau (pS422) comprises at least one or two or three or four or five or six HVRs selected from: (a) HVR-H1, comprising SEQ ID NO: 08 amino acid sequence; (b) HVR-H2; comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3, The amino acid sequence of SEQ ID NO: 15 is included.

In one embodiment, an antibody that specifically binds to human Tau (pS422) comprises at least one or two or three or four or five or six HVRs selected from: (a) HVR-H1, comprising SEQ ID NO: 08 amino acid sequence; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 05; and (f) HVR-L3, The amino acid sequence of SEQ ID NO: 15 is included.

In one embodiment, an antibody that specifically binds to human Tau (pS422) comprises at least one or two or three or four or five or six HVRs selected from: (a) HVR-H1, comprising SEQ ID NO: 08 amino acid sequence; (b) HVR-H2; comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3, Include SEQ ID NO: amino acid sequence of 15.

In one embodiment, the antibody that specifically binds to human Tau (pS422) comprises at least one, at least two or all three VH HVR sequences selected from the group consisting of i) (a) HVR-H1 comprising an amino acid sequence SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 18; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 10; or ii) (a HVR-H1 comprising an amino acid sequence of SEQ ID NO: 08; (b) HVR-H2 comprising an amino acid sequence of SEQ ID NO: 09; and (c) HVR-H3 comprising an amino acid sequence SEQ ID NO: 10.

In one embodiment, the antibody that specifically binds to human Tau (pS422) comprises i) (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 08; (b) HVR-H2 comprising an amine group Acid sequence SEQ ID NO: 18; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 10; or ii) (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 08; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 09; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 10.

In another embodiment, the antibody that specifically binds to human Tau (pS422) further comprises at least one, at least two or all three VL HVR sequences selected from the group consisting of i) (a) HVR-L1 comprising an amine group Acid sequence SEQ ID NO: 13; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 15; or ii) (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 12; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 05; and (c) HVR-L3 comprising an amine group Acid sequence SEQ ID NO: 15.

In another embodiment, the antibody that specifically binds to human Tau (pS422) comprises i) (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 13; (b) HVR- L2 comprising an amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising an amino acid sequence of SEQ ID NO: 15; or ii) (a) HVR-L1 comprising an amino acid sequence SEQ ID NO: 12; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 05; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 15.

In one embodiment, the antibody that specifically binds to human Tau (pS422) comprises i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2; ID NO: amino acid sequence of 18; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15, or ii) (a) HVR-H1 , which comprises the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2; comprising the amino acid sequence of SEQ ID NO: 09; (c) HVR-H3 comprising the amine of SEQ ID NO: a nucleic acid sequence; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 05; and (f) HVR- L3, which comprises the amino acid sequence of SEQ ID NO: 15, or iii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 08; (b) HVR-H2; comprising SEQ ID NO : amino acid sequence of 09; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; )HVR-L2 Comprising SEQ ID NO: 14 of the amino acid sequence; and (f) HVR-L3, which comprises SEQ ID NO: 15 of the amino acid sequence.

In another embodiment, the VH or VL of an antibody that specifically binds to human Tau (pS422) contains a substitution (eg, a conservative substitution), insertion or deletion relative to a reference sequence, but comprises an anti-human Tau (pS422) antibody of the sequence Retain the ability to bind to human Tau (pS422).

In another embodiment, the antibody that specifically binds to human Tau (pS422) is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the antibody that specifically binds to human Tau (pS422) is an antibody fragment, such as an Fv, Fab, Fab', scFv, CrossFab, scCrossFab, bifunctional antibody or F(ab') ₂ fragment. In another embodiment, the antibody that specifically binds to human Tau (pS422) is a full length antibody, eg, an intact IgGl or IgG4 antibody or other antibody type or isotype as defined herein.

In another embodiment, an antibody that specifically binds to human Tau (pS422) according to any of the above embodiments can be combined, either alone or in combination, in any combination, as described in Sections 1-5 below.

Antibody affinity

In certain embodiments, the dissociation constant (KD) of an antibody provided herein 100nM, 50 nM or between 1 nM and 100 nM (for example 10 ^-7 M or 10 ^-7 M or less, for example 10 ^-7 M to 10 ^-9 M).

In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, Fab-to-antigen is measured by balancing the Fab with a minimum concentration of ( ^125I )-labeled antigen in the presence of an unlabeled antigen titration series, followed by capture of the bound antigen with an anti-Fab antibody coated plate. The solution binds affinity (see, for example, Chen, Y. et al, J. Mol. Biol. 293 (1999) 865-881). In order to establish the analysis conditions, the multiwell plate MICROTITER ^® (Thermo Scientific) with 50mM sodium carbonate (pH 9.6) in the 5μg / ml of anti--Fab capture antibody (Cappel Labs) coated overnight, and then at room temperature (about 23 ℃) Block with 2% (w/v) bovine serum albumin in PBS for two to five hours. In a non-sorbent culture plate (Nunc #269620), mix 100 pM or 26 pM [ ¹²⁵ I] antigen with serially diluted Fab of interest (eg, consistent with evaluation of anti-VEGF antibody Fab-12, Presta, LG et al. , Cancer Res. 57 (1997) 4593-4599)). The Fab of interest is then incubated overnight; however, incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure equilibrium is achieved. Thereafter, the mixture is transferred to a capture plate at room temperature for incubation (eg, 1 hour). The solution was then removed and the dish was washed 8 times with PBS containing 0.1% polysorbate 20 (TWEEN-20®). When the disc has been dried, add 150μl per well of the scintillator ^{(MICROSCINT-20 TM; Packard)} , and counted on a TOPCOUNT ^TM plate counter (Packard) 10 min. Concentrations of each Fab providing less than or equal to 20% maximal binding were selected for competitive binding assays.

According to another embodiment, the use of BIACORE ^® surface plasmon resonance analysis method to measure the amount of Kd. For example, using BIACORE ^® -2000 or a ^{BIACORE ® -3000 (BIAcore, Inc.,} Piscataway, NJ), analyzed at approximately 10 response units (RU) at 25 deg.] C with immobilized antigen CM5 chip. In one embodiment, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxybutadiene are used according to the supplier's instructions. Amine (NHS) to activate a carboxymethylated dextran biosensor wafer (CM5, BIACORE, Inc.). The antigen was diluted to 5 μg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), followed by injection at a flow rate of 5 μl/min to obtain about 10 reaction units (RU) of the coupled protein. Following injection of the antigen, 1 M ethanolamine was injected to block unreacted groups. As regards the dynamic measurement, at 25 ℃, flow rate of about 25μl / min of injection containing 0.05% polysorbate 20 (TWEEN-20 ^TM) Fab PBS twice the surfactant (PBST) in serial dilutions ( 0.78nM to 500nM). Using simple one to one Langmuir binding model (one-to-one Langmuir binding model) (BIACORE ® Evaluation Software version 3.2), simultaneous fitting the association and to calculate the association rate solution (k _on) and dissociation from the sensor pattern rate (k _off ). The equilibrium dissociation constant (Kd) is calculated as the _koff / _kon ratio (see, for example, Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). By the above surface plasmon resonance analysis, if the association rate exceeds 10 ⁶ M ^-1 S ^-1 , the association rate can be measured using a fluorescence quenching technique, such as a spectrometer (such as having a stirring ratio) Measurement of PBS (pH 7.2) at 25 ° C in the presence of an increased concentration of antigen measured in a color tube equipped flow spectrometer (Aviv Instruments) or 8000-series SLM-AMINCO ^TM spectrometer (ThermoSpectronic) The increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab format).

2. Antibody fragment

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , CrossFab, scCrossFab, Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson, PJ et al, Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, for example, Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (ed.), Springer-Verlag, New York (1994), pp. 269-315; WO 93/16185; US 5,571,894 and US 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising rescue receptor binding epitope residues and increased in vivo half-life, see US 5,869,046.

A bifunctional antibody is an antibody fragment that has two antigen binding sites and can have bivalent or bispecificity. See, for example, EP 0 404 097; WO 1993/01161; Hudson, PJ et al, Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) )6444-6448. Trifunctional and tetrafunctional antibodies are also described in Hudson, P.J. et al., Nat. Med. 9 (20039 129-134).

A single domain antibody is an antibody fragment comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US 6,248,516).

Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

3. Humanized antibodies

Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibodies. Generally, a humanized antibody comprises one or more variable domains, wherein the HVR (eg, CDR) (or a portion thereof) is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies will also be included as appropriate Containing at least a portion of a full length human constant region. In some embodiments, some of the FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody from which the HVR residue is derived), eg, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their production are reviewed, for example, in Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and are further described, for example, in Riechmann, I. et al, Nature 332 (1988) 323. -329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; US 5,821,337, US 7,527,791, US 6,982,321 and US 7,087,409; Kashmiri, SV et al, Methods 36 (2005) 25-34 (Description of Specificity Determination Zone (SDR) Transplantation); Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (description "Surface Remodeling"); Dall'Acqua, WF et al., Methods 36 ( 2005) 43-60 (description "FR Reorganization"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 ( Describe the "guide selection" method for FR reorganization.

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, eg, Sims, MJ et al, J. Immunol. 151 (1993) 2296-2308); a framework region from a human antibody consensus sequence having a particular subgroup of light or heavy chain variable regions (see, eg, Carter, P. et al, Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Presta, LG et al, J. Immunol. 151 (1993) 2623-2632); human maturation (somatic mutation) framework regions or human germline framework regions (see for example Almagro, JC and Fransson, J., Front. Biosci. 13(2008) 1619-1633); and framework regions derived from screening FR libraries (see, for example, Baca, M. et al., J. Biol. Chem. 272 (1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (1996922611-22618).

4. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are singles that have binding specificities for at least two different sites Strain antibody. In certain embodiments, one of the binding specificities is directed against human Tau (pS422) and the other is directed against any other antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of human Tau (pS422). Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinantly co-presenting two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983). 537-540, WO 93/08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "杵-臼" engineering (see, for example, US 5,731,168). Multispecific antibodies can also be made by engineering engineered electrostatic steering effects of antibody Fc-heterodimeric molecules (WO 2009/089004); crosslinking two or more antibodies or fragments (see, for example, US 4,676,980 and Brennan) , M. et al., Science 229 (1985) 81-83); production of bispecific antibodies using leucine zippers (see, for example, Kostelny, SA et al, J. Immunol. 148 (1992) 1547-1553); A "bifunctional antibody" technique for obtaining bispecific antibody fragments (see, eg, Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (sFv) II Polymer (see, for example, Gruber, M et al, J. Immunol. 152 (1994) 5368-5374); and preparation of trispecific as described, for example, in Tutt, A. et al. J. Immunol. 147 (1991) 60-69. Sexual antibodies.

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, for example, US 2006/0025576).

The antibodies or fragments herein also include "double-effect Fab" or "DAF" comprising an antigen binding site that binds to human Tau (pS422) and another different antigen (see, for example, US 2008/0069820).

The antibodies or fragments herein also include the multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792 and WO 2010/145793.

5. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, with the proviso that the final construct possesses desirable characteristics, such as antigen binding.

a) substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Relevant sites for induction mutation induction include HVR and FR. Conservative substitutions are shown in the table under the heading "Better substitutions." Further substantial changes are provided in the table entitled "Exemplary Substitutions" and the reference amino acid side chain classes are further described below. Amino acid substitutions can be introduced into the relevant antibodies and screened for products having the desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Amino acids can be grouped according to the properties of the shared side chain: (1) hydrophobicity: n-leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidity: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions require members of one of these categories to be replaced by members of another category.

One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody (eg, a humanized antibody). In general, the resulting variants selected for further study are improved (eg, improved) in certain biological properties relative to the parent antibody (eg, increased affinity, reduced immunogenicity) and/or will remain substantially retained Certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody that can be conveniently produced, for example, using phage-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated and a variant antibody is presented on the phage and a variant antibody having a particular biological activity (eg, binding affinity) is screened.

Alterations (e.g., substitutions) can be made in the HVR, e.g., to improve antibody affinity. Residues that can be encoded in HVR "hot spots" (ie, codons encoded by codons that undergo high frequency mutations during somatic cell maturation (see, eg, Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196), and / Such alterations can be made with the resulting variant VH or VL of the test binding affinity in the residue of the contact antigen. By constructing and reselecting from a secondary library Affinity maturation techniques have been described, for example, in Hoogenboom, H. R. et al. Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation techniques, diversity can be introduced to a variable selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. In the gene. A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach in which several HVR residues (eg, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be identified one by one, for example, using alanine scanning mutation induction or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs as long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes in the HVR that do not substantially reduce binding affinity (eg, conservative substitutions as provided herein) can be performed in the HVR. Such alterations may, for example, be in addition to residues in the HVR that contact the antigen. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two or three amino acid substitutions.

A method suitable for identifying residues or regions that can be targeted for mutation-induced antibodies is referred to as "alanine scanning mutation induction" as described by Cunningham, BC and Wells, JA, Science 244 (1989) 1081-1085. Described. In this method, a residue or a set of target residues (eg, charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid ( For example, alanine or polyalanine) to determine if the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is utilized to identify the point of contact between the antibody and the antigen. Such contact residues and adjacent residues may be targets of substitution candidates or excluded from substitution candidates. Variants can be screened to determine if they contain the desired properties.

Amino acid sequence inserts include a residue from one residue to one hundred or more residues Amino and/or carboxyl terminal fusions of the polypeptides, as well as intrasequence inserts having single or multiple amino acid residues. Examples of terminal insertions include antibodies having N-terminal methionine residues. Other insertion variants of the antibody molecule include fusions of the N-terminus or C-terminus of the antibody with an enzyme (eg, for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

b) glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Addition of a glycosylation site to an antibody or deletion of a glycosylation site of an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

Where the antibody comprises an Fc region, the sugar attached thereto can be varied. Native antibodies produced by mammalian cells typically comprise a branched bi-antennary oligosaccharide, which is typically linked to the Asn297 of the CH2 domain of the Fc region by an N bond (see, for example, Wright, A. and Morrison, SL, TIBTECH 15 (1997)) 26-32). Oligosaccharides may include various carbohydrates such as mannose, N-ethyl glucosamine (GlcNAc), galactose and sialic acid, and fucose linked to GlcNAc in the "backbone" of the bi-touch oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to form antibody variants having certain improved properties.

c) Fc region variant

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. An Fc region variant can comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (eg, a substitution) at one or more amino acid positions.

In certain embodiments, the invention encompasses antibody variants having some, but not all, of the effector functions, which renders the antibody a candidate for application, in which antibody half-life is important in vivo, and some Effect functions such as complement and ADCC are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcyR binding ability (and thus may lack ADCC activity), but retains FcRn binding ability. The primary cells used to mediate ADCC, NK cells express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The performance of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in US 5,500,362 (see, for example, Hellstrom, I. et al, Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, a non-radioactive analysis methods (see, e.g. flow cytometry of cell-type non-radioactive cytotoxicity assays ACTI ^TM cells (CellTechnology, Inc.Mountain View, CA) ; and CytoTox 96 ^® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI )). Suitable effector cells for such analysis include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the relevant molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes, R. et al, Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assays can also be performed to confirm that the antibody does not bind to Clq and therefore does not have CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC analysis can be performed (see, for example, Gazzano-Santoro, H. et al, J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al, Blood 101 (2003) 1045-1052; Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo elimination rate/half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al, Int. Immunol. 18 (2006: 1759-1769).

Antibodies with reduced effector function include antibodies to one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US 6,737,056). Such Fc region Mutations include Fc region mutations having substitutions of two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc region mutations in which residues 265 and 297 are substituted with alanine. (US 7,332,581).

Certain antibody variants that are modified or reduced in combination with FcR are described (see, for example, US 6,737,056; WO 2004/056312; and Shields, R.L. et al, J. Biol. Chem. 276 (2001) 6591-6604).

In some embodiments, the alteration is made in an Fc region that alters (ie, decreases) C1q binding and/or complement dependent cytotoxicity (CDC), such as, for example, US 6,194,551, WO 99/51642, and Idusogie, EE, et al. J. Immunol. 164 (2000) 4178-4184.

The half-life is extended and is associated with the neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al, J. Immunol. 117 (1976) 587-593; and Kim, JK et al, J. Immunol. The combined improved antibody of 24 (1994) 2429-2434) is described in US 2005/0014934. These antibodies comprise an Fc region having one or more substitutions that modify the binding of the Fc region to FcRn. Such Fc region variants include variants having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, substitution of residue 434 of the Fc region (US 7,371,826).

See also Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351 for other examples of Fc region variants.

d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to produce a cysteine engineered antibody, such as a "thioMAb" wherein one or more residues of the antibody are substituted with a cysteine residue. In a particular embodiment, the substituted residue occurs at a reachable site of the antibody. By substituting their residues with cysteine, the reactive thiol group is thereby localized to the accessible site of the antibody And can be used to bind an antibody to other moieties, such as a drug moiety or a linker-drug moiety, to produce an immunoconjugate as further described herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU) of the heavy chain Fc region Numbering). The cysteine engineered antibody can be produced as described, for example, in US 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-protein portions known in the art and readily available. Portions suitable for derivatizing antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone , poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and Glycan or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polyoxypropylene/ethylene oxide copolymer, polyoxyethylene polyol (such as glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, the polymers can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be modified, whether the antibody derivative will be used in a given condition, etc. .

In another embodiment, a combination of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein portion is a carbon nanotube (Kam, N. W. et al, Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not damage the general cells but heat the non-protein portion to the temperature at which cells that kill the antibody-non-protein portion proximal.

D. Recombination methods and compositions

Antibodies can be produced using recombinant methods and compositions such as those described in U.S. Patent 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-human Tau (pS422) antibody described herein is provided. Such nucleic acids may encode a VL-containing amino acid sequence of an antibody and/or an amino acid sequence comprising VH (eg, a light chain and/or a heavy chain of an antibody). The nucleic acid can encode an amino acid sequence comprising a VL and/or an amino acid sequence comprising a VH of an antibody (eg, a light chain and/or a heavy chain of an antibody). In another embodiment, one or more vectors (eg, expression vectors) comprising the nucleic acid are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (eg, has been transformed as follows): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH, or (2) comprising a coding A first vector comprising a nucleic acid of an amino acid sequence of antibody VL, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising antibody VH. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (eg, Y0, NSO, Sp20 cells). In one embodiment, a method of making an anti-human Tau (pS422) antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above, and optionally from a host cell, under conditions suitable for expression of the antibody (or host cell culture medium) recover the antibody.

For recombinant production of an anti-human Tau (pS422) antibody, a nucleic acid encoding an antibody, eg, as described above, is isolated and inserted into one or more vectors for further colonization and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

Host cells suitable for the selection or expression of an antibody-encoding vector include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effect functions are not required. For the performance of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199 and US 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the performance of antibody fragments in E. coli. The soluble fraction can be isolated from the bacterial cell paste and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable for the selection or expression of hosts for antibody-encoding vectors, including glycosylation pathways that have been "humanized" such that the antibodies produced are partially or completely human. Glycosylated forms of fungi and yeast strains. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

Host cells suitable for the expression of glycosylated antibodies can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in combination with insect cells, specifically for transfecting Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example US 5,959,177, US 6,040,498, US 6,420,548 , US 7,125,978 and US 6,417,429 (produced as described PLANTIBODIES ^TM technology antibodies in transgenic plants genes colonization).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be suitable. Other examples of suitable mammalian host cell lines are the monkey kidney CV1 strain transformed with SV40 (COS-7); human embryonic kidney strains (e.g., for example in Graham, FL et al, J. Gen Virol. 36 (1977) 59-74. 293 or 293 cells described); baby hamster kidney cells (BHK); mouse sertoli cells (as for example in Mather, JP, Biol. Reprod. 23 (1980) 243-252 TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; Buffalo rat liver cells (buffalo rat Liver cell) (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); as for example in Mather, JP et al., Annals NYAcad. Sci. 383 (1982) TRI cells as described in 44-68; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub, G. et al., Proc. Natl) .Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for producing antibodies, see for example Yazaki, P. And Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

E. Analysis method

Anti-human Tau (pS422) antibodies, non-covalent complexes as reported herein, can be identified, screened or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.

1. Combining analysis and other analysis methods

The antigen-binding activity of an antibody that specifically binds to human Tau (pS422) is tested, for example, by a known method such as ELISA, αLISA, Western blot, antibody or reverse phase array.

In an exemplary ELISA or αLISA assay, Tau (pS422) in a solution (cell supernatant, cell or tissue lysate, body fluid, etc.) is bound by a capture antibody that specifically binds to Tau (pS422) An epitope or a conformation and a Tau (pS422) in the detection antibody that is coupled to the detection entity, the detection entity specifically binding to a second epitope or configuration of Tau (pS422). The readings are based on the detected entities (chemiluminescence, fluorescence, energy transfer induced luminescence, etc.). In some cases, the same antibody can be used in the same assay to capture and detect antibodies to detect aggregated forms of Tau (pS422) (see, for example, Tokuda, T. et al., Neurology 75 (2010) 1766-1772).

In the case of an antibody array, the antibody is spotted onto a glass or nitrocellulose wafer. The slides were blocked and incubated with a solution containing Tau (pS422), washed to remove unbound antibody and the bound antibody was detected with a fluorescently labeled corresponding secondary antibody. Fluorescent slide scanner Measure the fluorescent signal. Similarly for reverse phase arrays, recombinant Tau (pS422), cell supernatant, cell or tissue lysates, body fluids, and the like are spotted onto glass or nitrocellulose wafers. Slides were blocked and individual arrays were incubated with antibodies against specific epitopes on Tau (pS422). The unbound antibody is washed away and the bound antibody is detected with a fluorescently labeled corresponding secondary antibody. Fluorescent signals were measured by a fluorescent slide scanner (Dernick, G. et al., J. Lipid Res. 52 (2011) 2323-2331).

In an example of the Western blotting method, recombinant Tau (pS422) or Tau (pS422) derived from cell supernatant, cell or tissue lysate, body fluid, etc., is aggregated by molecular weight separation in SDS PAGE or natural gel conditions and Draw it onto a nitrocellulose or PVDF membrane. After blocking, the membrane is incubated with antibodies specific for the amino acid sequence or configuration of Tau (pS422). The membrane is then washed to remove unbound antibody. The bound antibody is detected by a corresponding secondary antibody coupled to a detection entity for chemiluminescence or fluorescence or other detection means. An antibody specific for the amino acid sequence of Tau (pS422) will bind to Tau (pS422) in various aggregated forms and thus have various molecular weights as long as the epitope is not blocked by aggregation. On the other hand, conformation-specific antibodies will only detect certain aggregated forms of Tau (pS422) that reveal bands only at specific molecular weights (see, for example, Towbin, H. et al., Proc. Natl. Acad. Sci. USA). 76 (1979) 4350-4353; Burnette, WN, Anal. Biochem. 112 (1981) 195-203).

2. Activity analysis

In one aspect, an assay for identifying a biologically active anti-human Tau (pS422) antibody is provided. Biological activity may include, for example, prevention/reduction/inhibition of Tau (pS422)-induced cytotoxicity, and/or prevention/reduction/inhibition of intercellular transmission of oligomeric human Tau (pS422), and/or reduction of Tau (pS422) in LUHMES cells. Induced caspase activity. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, the biological activity of an antibody of the invention is tested.

Secretion of Tau (pS422) by the addition of cells containing cell death leading to receptor neurons The modified medium is used to assess protective biological activity. This toxicity can be reversed by the addition of a protective antibody as described herein. The toxicity of secreted Tau (pS422) has previously been determined (Emmanouilidou, E. et al., J. Neurosci., 30 (2010) 6838-6851).

F. Pharmaceutical formulations

By mixing such non-covalent complexes of the desired purity with one or more pharmaceutically acceptable carriers selected as appropriate (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. (ed.) (1980)) A pharmaceutical formulation of a non-covalent complex as described herein in the form of a lyophilized formulation or an aqueous solution is prepared. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including Ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexahydrochloroammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzene Methanol; alkyl paraben, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; a molecular weight (less than about 10 residues) polypeptide; a protein such as serum albumin, gelatin or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamic acid, Asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol , trehalose or sorbitol; salt-forming ions, such as sodium; metal mismatch (E.g., zinc - compound wrong protein); and / or non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as the human soluble PH-20 hyaluronan glycoprotein, such as rHuPH20 (HYLENEX) ®, Baxter International, Inc.). Certain exemplary sHASEGPs (including rhuPH20) and methods of use are described in US 2005/0260186 and US 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006/044908, and the formulations described in WO 2006/044908 include histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. These active ingredients are suitably present in combination in an amount effective to achieve the intended purpose.

The active ingredient may be coated in microcapsules (for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, prepared by coacervation techniques or by interfacial polymerization; colloidal state In drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.

Formulations intended for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

G. Methods and compositions

Any non-covalent complex as reported herein can be used in a method of treatment.

In one aspect, a non-covalent complex as reported herein for use as a medicament is provided. In other aspects, a non-covalent complex as reported herein for use in treating Alzheimer's disease is provided. In certain embodiments, non-covalent complexes as reported herein for use in a method of treatment are provided. In certain embodiments, the invention provides a non-covalent complex as reported herein for use in a method of treating an individual having Alzheimer's disease, the method comprising administering to the individual an effective amount of non-covalent Complex. In one such embodiment, the method additionally comprises administering to the individual an effective amount of at least one other therapeutic agent, for example, as described below. In other embodiments, the invention provides a non-covalent complex for use in inhibiting human gods Tau (pS422)-induced cytotoxicity in menstrual cells and glial cells. In certain embodiments, the present invention provides a non-covalent complex for use in a method of inhibiting Tau (pS422)-induced cytotoxicity in human neurons and glial cells in an individual, the method comprising administering to an individual Non-covalent complexes to inhibit Tau (pS422)-induced cytotoxicity in human neurons and glial cells. The "individual" according to any of the above embodiments is preferably a human.

In another aspect, the invention provides the use of a non-covalent complex as reported herein for the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating Alzheimer's disease. In another embodiment, the medicament is for use in a method of treating Alzheimer's disease, the method comprising administering to an individual having Alzheimer's disease an effective amount of an agent. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent, such as described below. In another embodiment, the agent is for inhibiting Tau (pS422)-induced cytotoxicity in human neurons and glial cells. In another embodiment, the agent is for use in a method of inhibiting Tau (pS422)-induced cytotoxicity in human neurons and glial cells in an individual, the method comprising administering to the individual an effective amount of an agent to inhibit human neurons And Tau (pS422)-induced cytotoxicity in glial cells. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides a method of treating Alzheimer's disease. In one embodiment, the method comprises administering to an individual having such Alzheimer's disease an effective amount of a non-covalent complex as reported herein. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent as described below. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides a method for inhibiting Tau (pS422)-induced cytotoxicity in human neurons and glial cells in an individual. In one embodiment, the method comprises administering to the individual an effective amount of a non-covalent complex as reported herein to inhibit Tau (pS422)-induced cytotoxicity in human neurons and glial cells. In a In the examples, the "individual" is a human.

In another aspect, the invention provides a pharmaceutical formulation comprising any non-covalent complex as reported herein, for example, for use in any of the above methods of treatment. In one embodiment, the pharmaceutical formulation comprises any non-covalent complex as reported herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any non-covalent complex as reported herein and at least one additional therapeutic agent, such as described below.

The antibodies of the invention may be used in therapy alone or in combination with other agents. For example, an antibody of the invention can be administered co-administered with at least one other therapeutic agent.

Such combination therapies mentioned above encompass combination administration (in which two or more therapeutic agents are included in the same or separate formulations) and administered separately, in which case the administration of the antibodies of the invention may be Performed before, concurrently with, and/or after administration of the additional therapeutic agent. In one embodiment, the administration of the non-covalent complex and the administration of the additional therapeutic agent are within about one month of each other; or within about one, two, or three weeks; or about one, two, and three days In four days, five days or six days.

Non-covalent complexes (and any additional therapeutic agents) as reported herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as topical treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Partially, depending on the short-term or long-term nature of administration, administration can be by any suitable route (e.g., by injection, such as intravenous or subcutaneous injection). Various administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at various time points, bolus administration, and pulsed infusion.

Non-covalent complexes as reported herein will be formulated, administered, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the agent, the method of administration, the time course of administration, and other known to the practitioner. factor. Non-covalent complexes need not be, but may optionally be, one or more drugs currently used to prevent or treat the condition The agents are formulated together. The effective amount of such other agents will depend on the amount of non-covalent complex present in the formulation, the type of condition or treatment, and other factors as described above. Such agents are typically administered in the same dosages and using the routes of administration described herein, or at about 1% to 99% of the dosages described herein, or in any dosage and empirically/clinically determined to be appropriate. use.

For the prevention or treatment of a disease, an appropriate dose of a non-covalent complex as reported herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of non-covalent complex, The severity and duration of the disease, whether the non-covalent complex is administered for prophylactic or therapeutic purposes, prior therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The non-covalent complex is appropriately administered to the patient once or over a series of treatments. Whether administered by one or more separate administrations, or by continuous infusion, depending on the type and severity of the disease, the initial candidate dose for administration of the antibody to the patient is, for example, from about 1 [mu]g/kg to 15 mg/kg (eg, 0.5 mg/). Kg-10mg/kg). A typical daily dose may range from about 1 [mu]g/kg to 100 mg/kg or higher, depending on the factors mentioned above. For repeated administration over several days or longer, treatment depending on the condition usually lasts until the symptoms of the disease are inhibited. An exemplary dosage of the non-covalent complex will range from about 0.05 mg/kg to about 50 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 15 mg/kg, 25 mg/mg, or 50 mg/kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example, every other week or every other three weeks (e.g., such that the patient receives about two to about twenty or, for example, about six doses of antibody). The initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be suitable. The course of this therapy is easily monitored by conventional techniques and analysis.

It will be appreciated that any of the above methods of formulation or treatment may be carried out using the immunoconjugates of the invention in place of or in addition to the covalent complex.

III. Article manufacturing

In another aspect of the invention, an article of manufacture comprising a substance useful for treating, preventing, and/or diagnosing a condition described above is provided. The article comprises a container and a label or package insert attached to the container or attached to the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers may be formed from a variety of materials such as glass or plastic. The container may be formed from a variety of materials such as glass or plastic. The container holds a separate composition or a combination with another composition effective to treat, prevent, and/or diagnose a condition, and may have a sterile access port (eg, the container may be a vein having a stopper pierceable by a hypodermic needle) Inside solution bag or vial). At least one active agent in the composition is an antibody of the invention. The label or internal page instructions indicate that the composition is used to treat a selected condition. Additionally, the article of manufacture may comprise (a) a first container comprising the composition, wherein the composition comprises an antibody of the invention; and (b) a second container comprising the composition, wherein the composition comprises another cytotoxicity Agent or therapeutic agent. Articles in embodiments of the invention may additionally include an in-page specification indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article may additionally comprise a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution) Two (or third) containers. It may additionally include other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It is to be understood that any of the above products may comprise, in addition to or in addition to an anti-human Tau (pS422) antibody, an immunoconjugate of the invention.

IV. Specific Embodiments

A non-covalent complex that specifically binds to a haptenated antibody of human Tau (pS422) and an anti-blood brain barrier receptor/hapten bispecific antibody.

2. A non-covalent complex comprising a haptenated antibody and a bispecific antibody that specifically binds to human Tau (pS422), the bispecific antibody specifically binding to human Tau (pS422) specifically binding The first binding specificity and specificity of the hapten of the haptenated antibody binds to a second binding specificity of a blood brain barrier receptor, wherein the specific knot The haptenated antibody that binds to human Tau (pS422) specifically binds by the first binding specificity of the bispecific antibody.

3. The non-covalent complex according to any one of embodiments 1 to 2, wherein the haptenized anti-system specifically binding to human Tau (pS422) is selected from the group consisting of: specifically binding to human Tau ( a biotinylated antibody of pS422), a theophylline antibody that specifically binds to human Tau (pS422), a digoxigenin antibody that specifically binds to human Tau (pS422), and a carbon that specifically binds to human Tau (pS422) Boronated antibodies, luciferylated antibodies that specifically bind to human Tau (pS422), helical antibodies that specifically bind to human Tau (pS422), and bromodeoxyuridine that specifically binds to human Tau (pS422) antibody.

4. The non-covalent complex according to any one of embodiments 1 to 3, wherein the blood-brain barrier receptor system is selected from the group consisting of: transferrin receptor (TfR), insulin receptor, insulin-like growth factor Receptor (IGF receptor), low density lipoprotein receptor-related protein 8 (LRP8), low density lipoprotein receptor-related protein 1 (LRP1), and heparin-binding epidermal growth factor-like growth factor (HB-EGF).

5. The non-covalent complex of any of embodiments 1 to 4, wherein the bispecific antibody is a full length antibody comprising two binding sites.

6. The non-covalent complex of any one of embodiments 1 to 5, wherein the bispecific antibody is one that has been fused to one or two scFv or scFab or CrossFab or scCrossFab and comprises three or four binding sites Full length antibody.

The non-covalent complex according to any one of embodiments 1 to 6, wherein the bispecific anti-system is selected from the group consisting of an antibody fragment, F(ab')2 and a bifunctional antibody.

The non-covalent complex of any one of embodiments 1 to 7, wherein the bispecific antibody is a humanized or human antibody.

The non-covalent complex of any one of embodiments 1 to 8, wherein the bispecific antibody does not contain an effector function.

10. The non-covalent composite according to any one of embodiments 1 to 9, wherein in the embodiment Bispecific antibodies do not have a functional Fc region.

The non-covalent complex of any one of embodiments 1 to 10, wherein the bispecific antibody does not have an Fc region.

The non-covalent complex according to any one of embodiments 1 to 12, wherein the bispecific antibody has an Fc region comprising a human IgG1 subclass of the mutations L234A, L235A and P329G, wherein the positions are according to the Kabat Fc region The number (Kabat EU index) is determined.

The non-covalent complex according to any one of embodiments 1 to 12, wherein the bispecific antibody has an Fc region comprising a human IgG4 subclass of the mutations S228P, L235E and P329G, wherein the positions are according to the Kabat Fc region The number (Kabat EU index) is determined.

The non-covalent complex of any one of embodiments 1 to 13, wherein the bispecific antibody comprises a) a binding site for a hapten that specifically binds to a haptenated antibody of human Tau (pS422) and a binding site for a blood brain barrier receptor, or b) two binding sites for a hapten that specifically binds to a haptenated antibody of human Tau (pS422) and a binding site for a blood brain barrier receptor, or c) a binding site for a hapten that specifically binds to a haptenized antibody of human Tau (pS422) and two binding sites for a blood brain barrier receptor, or d) two binding sites for haptens that specifically bind to the haptenated antibody of human Tau (pS422) and two binding sites for the blood brain barrier receptor, Wherein in the case of b) and c), one of the heavy chains of the bispecific antibody comprises a purine mutation and each of the other strands comprises a purine mutation.

The non-covalent complex according to any one of embodiments 1 to 14, wherein the bispecific antibody comprises two binding sites for a hapten that specifically binds to a haptenized antibody of human Tau (pS422) And two binding sites for the blood-brain barrier receptor.

16. The non-covalent complex of any one of embodiments 1 to 15, wherein the bispecific antibody has a haptenated antibody that specifically binds to human Tau (pS422). Two binding specificities of haptens (two anti-hapten binding specificities) and specific binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificities) or low density Two binding specificities of lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor-related protein 8 binding specificity).

The non-covalent complex according to any one of embodiments 1 to 16, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody which specifically binds to human Tau (pS422) is an antibody heavy A strand variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 65, (b) comprising a heavy chain CDR2 of the amino acid sequence SEQ ID NO: (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 67, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 69, and (e) comprises the amino acid sequence SEQ ID NO: 70 The light chain CDR2, and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO:71.

The non-covalent complex according to any one of embodiments 1 to 17, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody which specifically binds to human Tau (pS422) is humanized Binding specificity.

19. The non-covalent complex according to any one of embodiments 1 to 18, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody which specifically binds to human Tau (pS422) comprises, as embodied The CDRs and acceptor human frameworks in Example 17 (e.g., human immunoglobulin framework or human consensus framework).

The non-covalent complex according to any one of embodiments 1 to 19, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody which specifically binds to human Tau (pS422) is an antibody heavy A chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 73, (b) comprising a heavy chain CDR2 of the amino acid sequence SEQ ID NO: (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 75, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 77, and (e) comprises the amino acid sequence SEQ ID NO: 78 Light chain CDR2, and (f) comprising the amino acid sequence SEQ ID NO: 79 light chain CDR3.

The non-covalent complex according to any one of embodiments 1 to 20, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody which specifically binds to human Tau (pS422) is an antibody heavy a chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, and the amino acid sequence SEQ ID NO: 68 or 76, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequence.

The non-covalent composite according to any one of embodiments 1 to 21, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 A %-consistent VH sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-digoxigenin antibody comprising the sequence retains the ability to bind to digoxin. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 68 or 76 have been substituted, inserted, and/or deleted.

The non-covalent complex of any one of embodiments 1 to 22, wherein the substitution, insertion or deletion is present in a region outside the CDR (i.e., in the FR).

The non-covalent complex of any one of embodiments 1 to 23, wherein the digoxigenin binding specificity comprises the VH sequence of SEQ ID NO: 68 or 76, including post-translational modifications of the sequence.

The non-covalent complex according to any one of embodiments 1 to 24, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody which specifically binds to human Tau (pS422) is an antibody heavy A chain variable domain and an antibody light chain variable domain pair additionally comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with the amino acid sequence SEQ ID NO: 72 or 80 , 97%, 98%, 99% or 100% sequence identity of the light chain variable domain (VL).

26. The non-covalent complex of any of embodiments 1 to 25, wherein there is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 A %-consistent VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-digoxigenin antibody comprising the sequence retains the ability to bind to digoxin. In some embodiments In the SEQ ID NO: 72 or 80, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted.

27. The non-covalent complex of any of embodiments 1 to 26, wherein the substitution, insertion or deletion is present in a region outside the CDR (i.e., in the FR).

The non-covalent complex of any one of embodiments 1 to 27, wherein the digoxigenin binding specificity comprises the VL sequence of SEQ ID NO: 72 or 80, including post-translational modifications of the sequence.

The non-covalent complex of any one of embodiments 1 to 16, wherein the bispecific antibody comprises a first binding to biotin that specifically binds to a biotinylated antibody that specifically binds to human Tau (pS422) Specificity (anti-biotin binding specificity; anti-BI binding specificity) and specific binding to (human) transferrin receptor (anti-(human) transferrin receptor binding specificity; anti-(h)TfR binding Specificity) or second binding specificity of low density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor-related protein 8 binding specificity; anti-LRP8 binding specificity).

The non-covalent complex of any one of embodiments 1 to 16 and 29, wherein the bispecific antibody has two binding specificities that specifically bind to a biotinylated payload (two avidin bindings) Specificity and specific binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor) The two binding specificities of the relevant protein 8 binding specificity).

The non-covalent complex according to any one of embodiments 1 to 16 and 29 to 30, wherein the binding specificity of the biotin which specifically binds to the biotinylated antibody which specifically binds to human Tau (pS422) is An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 81, (b) comprising the amino acid sequence SEQ ID NO: 82 The chain CDR2, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 83, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 85, and (e) comprises the amino acid sequence SEQ ID NO : 86 light chain CDR2, and (f) comprising the amino acid sequence SEQ ID NO: 87 Chain CDR3.

The non-covalent complex according to any one of embodiments 1 to 16 and 29 to 31, wherein the binding specificity of the biotin which specifically binds to the biotinylated antibody which specifically binds to human Tau (pS422) is Humanization combined with specificity.

The non-covalent complex according to any one of embodiments 1 to 16 and 29 to 32, wherein the binding specificity of the biotin which specifically binds to the biotinylated antibody which specifically binds to human Tau (pS422) comprises The CDRs and acceptor human frameworks as in Example 31 (e.g., human immunoglobulin framework or human consensus framework).

The non-covalent complex according to any one of embodiments 1 to 16 and 29 to 33, wherein the binding specificity of the biotin which specifically binds to the biotinylated antibody which specifically binds to human Tau (pS422) is An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 89, (b) comprising an amino acid sequence of SEQ ID NO: 90 The chain CDR2, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 91, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 93, and (e) comprises the amino acid sequence SEQ ID NO The light chain CDR2 of 94, and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO:95.

The non-covalent complex according to any one of embodiments 1 to 16 and 29 to 34, wherein the binding specificity of the biotin which specifically binds to the biotinylated antibody which specifically binds to human Tau (pS422) is An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96 with the amino acid sequence SEQ ID NO: 84 or 92 %, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequences.

The non-covalent composite according to any one of embodiments 1 to 16 and 29 to 35, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% consensus VH sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-biotin antibody comprising the sequence retains the ability to bind to biotin.

The non-covalent complex of any one of embodiments 1 to 16 and 29 to 36, wherein a total of 1 to 10 amino acids of SEQ ID NO: 84 or 92 have been substituted, inserted and/or deleted.

The non-covalent complex of any one of embodiments 1 to 16 and 29 to 37, wherein the substitution, insertion or deletion is present in a region outside the CDR (i.e., in the FR).

The non-covalent complex of any one of embodiments 1 to 16 and 29 to 38, wherein the biotin binding specificity comprises the VH sequence of SEQ ID NO: 84 or 92, including post-translational modifications of the sequence .

The non-covalent complex according to any one of embodiments 1 to 16 and 29 to 39, wherein the binding specificity of the biotin which specifically binds to the biotinylated antibody which specifically binds to human Tau (pS422) is An antibody heavy chain variable domain and an antibody light chain variable domain pair additionally comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence SEQ ID NO: 88 or 96, 96%, 97%, 98%, 99% or 100% sequence identity of the light chain variable domain (VL).

41. The non-covalent complex of any of embodiments 1 to 16 and 29 to 40, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% identity VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-biotin antibody comprising the sequence retains the ability to bind to biotin.

The non-covalent complex of any one of embodiments 1 to 16 and 29 to 41, wherein a total of 1 to 10 amino acids in SEQ ID NO: 88 or 96 have been substituted, inserted and/or deleted.

The non-covalent complex of any one of embodiments 1 to 16 and 29 to 42, wherein the substitution, insertion or deletion is present in a region outside the CDR (i.e., in the FR).

The non-covalent complex of any one of embodiments 1 to 16 and 29 to 43, wherein the biotin binding specificity comprises the VL sequence of SEQ ID NO: 88 or 96, including post-translational modifications of the sequence.

The non-covalent complex of any one of embodiments 1 to 16, wherein the bispecific antibody comprises a first binding specificity that specifically binds to the theophylline payload (anti-theophylline binding Heterologous; anti-THEO binding specificity) and specific binding to (human) transferrin receptor (anti-(human) transferrin receptor binding specificity; anti-(h)TfR binding specificity) or low density lipoprotein The second binding specificity of receptor-associated protein 8 (anti-low-density lipoprotein receptor-related protein 8 binding specificity; anti-LRP8 binding specificity).

The non-covalent complex according to any one of embodiments 1 to 16 and 45, wherein the bispecific antibody has two binding specificities for specific binding to the theophylline payload (two anti-theophylline binding specificities) And specific binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor-related) Two binding specificities of protein 8 binding specificity).

The non-covalent complex according to any one of embodiments 1 to 16 and 45 to 46, wherein the binding specificity of the theophylline which specifically binds to the theophylline antibody which specifically binds to human Tau (pS422) is resistant a heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 97, (b) a heavy chain comprising the amino acid sequence SEQ ID NO: 98 CDR2, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 99, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 101, and (e) comprises the amino acid sequence SEQ ID NO: The light chain CDR2 of 102 and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO:103.

The non-covalent complex according to any one of embodiments 1 to 16 and 45 to 47, wherein the binding specificity of theophylline which specifically binds to the theophylline antibody which specifically binds to human Tau (pS422) is Humanization combined with specificity.

The non-covalent complex according to any one of embodiments 1 to 16 and 45 to 48, wherein the binding specificity of the theophylline which specifically binds to the theophylline antibody which specifically binds to human Tau (pS422) comprises The CDRs and acceptor human frameworks as in Example 47 (e.g., human immunoglobulin framework or human consensus framework).

The non-covalent complex according to any one of embodiments 1 to 16 and 45 to 49, wherein the binding to the theophylline which specifically binds to the theophylline antibody which specifically binds to human Tau (pS422) Specificity is an antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 105, (b) comprising an amino acid sequence SEQ ID NO: The heavy chain CDR2 of 106, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 107, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 109, and (e) comprises the amino acid sequence The light chain CDR2 of SEQ ID NO: 110 and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO: 111.

The non-covalent complex according to any one of embodiments 1 to 16 and 45 to 50, wherein the binding specificity of the theophylline which specifically binds to the theophylline antibody which specifically binds to human Tau (pS422) is An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96 with the amino acid sequence SEQ ID NO: 100 or 108 %, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequences.

The non-covalent composite according to any one of embodiments 1 to 16 and 45 to 51, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% consensus VH sequence contains a substitution (e.g., a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-theaine antibody comprising the sequence retains the ability to bind to theophylline.

The non-covalent complex of any one of embodiments 1 to 16 and 45 to 52, wherein a total of 1 to 10 amino acids in SEQ ID NO: 100 or 108 have been substituted, inserted and/or deleted.

The non-covalent complex of any one of embodiments 1 to 16 and 45 to 53, wherein the substitution, insertion or deletion is present in a region outside the CDR (i.e., in the FR).

The non-covalent complex of any one of embodiments 1 to 16 and 45 to 54, wherein the theophylline binding specificity comprises the VH sequence of SEQ ID NO: 100 or 108, including post-translational modifications of the sequence.

The non-covalent complex according to any one of embodiments 1 to 16 and 45 to 55, wherein the binding specificity of theophylline which specifically binds to the theophylline antibody which specifically binds to human Tau (pS422) is Antibody heavy chain variable domain and antibody light chain variable domain pair, additionally comprising an amino acid Sequence SEQ ID NO: 104 or 112 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of light chain variable Domain (VL).

57. The non-covalent complex of any of embodiments 1 to 16 and 45 to 56, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% identity VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-theaine antibody comprising the sequence retains the ability to bind to theophylline.

The non-covalent complex of any one of embodiments 1 to 16 and 45 to 57, wherein a total of 1 to 10 amino acids in SEQ ID NO: 104 or 112 have been substituted, inserted and/or deleted.

The non-covalent complex of any one of embodiments 1 to 16 and 45 to 58, wherein the substitution, insertion or deletion is present in a region outside the CDR (i.e., in the FR).

The non-covalent complex of any one of embodiments 1 to 16 and 45 to 59, wherein the theophylline binding specificity comprises the VL sequence of SEQ ID NO: 104 or 112, including post-translational modifications of the sequence.

The non-covalent complex of any one of embodiments 1 to 16, wherein the bispecific antibody comprises a first binding specificity (anti-luciferin binding specificity) that specifically binds to a fluoresceinated payload; Anti-FLUO binding specificity) and specific binding to (human) transferrin receptor (anti-(human) transferrin receptor binding specificity; anti-(h)TfR binding specificity) or low density lipoprotein receptor Second binding specificity of related protein 8 (anti-low density lipoprotein receptor associated protein 8 binding specificity; anti-LRP8 binding specificity).

The non-covalent complex of any one of embodiments 1 to 16 and 61, wherein the bispecific antibody has two binding specificities (two anti-fluoresceins) that specifically bind to the fluoresceinated payload Binding specificity and specific binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein Two binding specificities of the body-associated protein 8 binding specificity).

The non-covalent complex according to any one of embodiments 1 to 16 and 61 to 62, wherein the binding specificity to luciferin which specifically binds to a luciferylated antibody which specifically binds to human Tau (pS422) An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 113, (b) comprising an amino acid sequence SEQ ID NO: 114 The heavy chain CDR2, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 115, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 117, and (e) comprises the amino acid sequence SEQ The light chain CDR2 of ID NO:118 and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO:119.

The non-covalent complex according to any one of embodiments 1 to 16 and 61 to 63, wherein the binding specificity to luciferin which specifically binds to a luciferylated antibody which specifically binds to human Tau (pS422) Sex is the binding specificity of humanization.

The non-covalent complex according to any one of embodiments 1 to 16 and 61 to 64, wherein the binding specificity to luciferin which specifically binds to a luciferylated antibody which specifically binds to human Tau (pS422) Sexually comprises the CDRs as described in Example 63 and the acceptor human framework (e.g., human immunoglobulin framework or human consensus framework).

The non-covalent complex according to any one of embodiments 1 to 16 and 61 to 65, wherein the binding specificity to luciferin which specifically binds to a luciferylated antibody which specifically binds to human Tau (pS422) Is an antibody heavy chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96 with the amino acid sequence SEQ ID NO: 116. %, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequences.

The non-covalent composite according to any one of embodiments 1 to 16 and 61 to 66, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% consensus VH sequence contains a substitution (eg, a conservative substitution), insertion or deletion relative to a reference sequence, but the anti-luciferin antibody comprising the sequence retains the ability to bind to luciferin.

The non-covalent complex of any one of embodiments 1 to 16 and 61 to 67, wherein a total of 1 to 10 amino acids in SEQ ID NO: 116 have been substituted, inserted and/or deleted.

The non-covalent complex of any one of embodiments 1 to 16 and 61 to 68, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

The non-covalent complex of any one of embodiments 1 to 16 and 61 to 69, wherein the luciferin binding specificity comprises the VH sequence of SEQ ID NO: 116, including post-translational modifications of the sequence.

The non-covalent complex according to any one of embodiments 1 to 16 and 61 to 70, wherein the binding specificity to luciferin which specifically binds to a luciferylated antibody which specifically binds to human Tau (pS422) Is an antibody heavy chain variable domain and an antibody light chain variable domain pair additionally comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence SEQ ID NO: 96%, 97%, 98%, 99% or 100% sequence identity of the light chain variable domain (VL).

The non-covalent composite according to any one of embodiments 1 to 16 and 61 to 71, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% identity VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-luciferin antibody comprising the sequence retains the ability to bind to luciferin.

The non-covalent complex of any one of embodiments 1 to 16 and 61 to 72, wherein a total of 1 to 10 amino acids in SEQ ID NO: 120 have been substituted, inserted and/or deleted.

The non-covalent complex of any one of embodiments 1 to 16 and 61 to 73, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

The non-covalent complex of any one of embodiments 1 to 16 and 61 to 74, wherein the luciferin binding specificity comprises the VL sequence of SEQ ID NO: 120, including post-translational modifications of the sequence.

The non-covalent complex of any one of embodiments 1 to 16, wherein the bispecific antibody comprises a first binding specificity (anti-bromodeoxyuridine) that specifically binds to a net load of bromodeoxyuridine Binding specificity; anti-BrdU binding specificity) and specific binding to (human) transferrin receptor (anti-(human) transferrin receptor binding specificity; anti-(h)TfR binding specificity) Or a second binding specificity of low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor-related protein 8 binding specificity; anti-LRP8 binding specificity).

77. The non-covalent complex of any one of embodiments 1 to 16 and 76, wherein the bispecific antibody has two binding specificities that specifically bind to a net load of bromodeoxyuridine (two anti-bromine) Deoxyuridine binding specificity) and specific binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (low anti-low Two binding specificities of density lipoprotein receptor-related protein 8 binding specificity).

The non-covalent complex according to any one of embodiments 1 to 16 and 76 to 77, wherein the bromodeoxyuridine which specifically binds to a bromodeoxyuridine antibody which specifically binds to human Tau (pS422) The binding specificity of the glycoside is an antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 121, (b) comprising an amino acid sequence SEQ ID NO: 123 heavy chain CDR2, (c) comprising the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 125, (d) comprising the amino acid sequence SEQ ID NO: 126, the light chain CDR1, (e) comprising an amine The light chain CDR2 of SEQ ID NO: 127 and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO: 128.

The non-covalent complex according to any one of embodiments 1 to 16 and 76 to 78, wherein the bromodeoxyuridine which specifically binds to a bromodeoxyuridine antibody which specifically binds to human Tau (pS422) The binding specificity of the glycoside is humanized binding specificity.

The non-covalent complex according to any one of embodiments 1 to 16 and 76 to 79, wherein the bromodeoxyuridine which specifically binds to a bromodeoxyuridine antibody which specifically binds to human Tau (pS422) The binding specificity of the glycoside comprises the CDRs as described in the above examples and the acceptor human framework (e.g., human immunoglobulin framework or human common framework).

The non-covalent complex according to any one of embodiments 1 to 16 and 76 to 80, wherein the bromodeoxyuridine which specifically binds to a bromodeoxyuridine antibody which specifically binds to human Tau (pS422) The binding specificity of the glycoside is an antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 121 or 122, (b) comprising an amino acid The heavy chain CDR2 of SEQ ID NO: 123 or 124, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 125, and (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 126, ( e) a light chain CDR2 comprising the amino acid sequence SEQ ID NO: 127 and (f) a light chain CDR3 comprising the amino acid sequence SEQ ID NO: 128.

The non-covalent complex according to any one of embodiments 1 to 16 and 76 to 81, wherein the bromodeoxyuridine which specifically binds to a bromodeoxyuridine antibody which specifically binds to human Tau (pS422) The binding specificity of the glycoside is an antibody heavy chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94 with the amino acid sequence SEQ ID NO: 129 or 131. %, 95%, 96%, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequences.

The non-covalent composite according to any one of embodiments 1 to 16 and 76 to 82, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% consensus VH sequence contains a substitution (e.g., a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-bromo-deoxyuridine antibody comprising the sequence retains the ability to bind to bromodeoxyuridine.

The non-covalent complex of any one of embodiments 1 to 16 and 76 to 83, wherein a total of 1 to 10 amino acids in SEQ ID NO: 129 or 131 have been substituted, inserted and/or deleted.

The non-covalent complex of any one of embodiments 1 to 16 and 76 to 84, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

The non-covalent complex of any one of embodiments 1 to 16 and 76 to 85, wherein the bromodeoxyuridine binding specificity comprises the VH sequence of SEQ ID NO: 129 or 131, including translation of the sequence Post-modification.

The non-covalent complex according to any one of embodiments 1 to 16 and 76 to 86, wherein the bromodeoxyuridine which specifically binds to a bromodeoxyuridine antibody which specifically binds to human Tau (pS422) The binding specificity of the glycoside is an antibody heavy chain variable domain and an antibody light chain variable domain pair, which additionally Included with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence SEQ ID NO: 130 or 132 Light chain variable domain (VL).

The non-covalent composite of any one of embodiments 1 to 16 and 76 to 87, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% consensus VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-bromodeoxyuridine antibody comprising the sequence retains the ability to bind to bromodeoxyuridine.

The non-covalent complex of any one of embodiments 1 to 16 and 76 to 88, wherein a total of 1 to 10 amino acids of SEQ ID NO: 130 or 132 have been substituted, inserted and/or deleted.

The non-covalent complex of any of embodiments 1 to 16 and 76 to 89, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

The non-covalent complex of any one of embodiments 1 to 16 and 76 to 90, wherein the bromodeoxyuridine binding specificity comprises the VL sequence of SEQ ID NO: 130 or 132, including translation of the sequence Post-modification.

The non-covalent complex of any one of embodiments 1 to 91, wherein the haptenated antibody that specifically binds to human Tau (pS422) is in a hapten and an antibody that specifically binds to human Tau (pS422) Contains a linking group.

93. The non-covalent complex of embodiment 92, wherein the linking group is a peptide linking group.

94. The non-covalent complex of embodiment 92, wherein the linking group is a chemical linking group (non-peptide linking group).

The non-covalent complex of any one of embodiments 1 to 94, wherein the antibody that specifically binds to human Tau (pS422) is a full length antibody.

96. The non-covalent complex of any of embodiments 1 to 95, wherein the specific binding Antibody i) to human Tau (pS422) specifically binds to a polypeptide having the amino acid sequence SEQ ID NO: 03, and/or ii) does not bind to full length human Tau at 1 μg/mL (SEQ ID NO: 01) And/or iii) full length human Tau (SEQ ID NO: 02), and/or iv) that specifically binds to the phosphorylation at serine at position 422, specifically binds to the serine at position 422 Aggregates of phosphorylated human Tau (SEQ ID NO: 02), and/or v) specifically bind to human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A.

97. The non-covalent complex of any one of embodiments 1 to 96, wherein the antibody that specifically binds to human Tau (pS422) comprises a) in the heavy chain variable domain, SEQ ID NOs: 08, 18 and HVR of 10, or b) HVR of SEQ ID NOS: 08, 09 and 10 in the heavy chain variable domain.

The non-covalent complex of any one of embodiments 1 to 97, wherein the antibody that specifically binds to human Tau (pS422) additionally comprises a) in the light chain variable domain, SEQ ID NO: 13, 14 And HVR of 15 or b) HVR of SEQ ID NOS: 12, 05 and 15 in the light chain variable domain.

The non-covalent complex of any one of embodiments 1 to 98, wherein the antibody that specifically binds to human Tau (pS422) comprises a) in the heavy chain variable domain, SEQ ID NOs: 08, 18 and HVR of 10, and HVR of SEQ ID NOS: 13, 14 and 15 in the light chain variable domain, or b) HVR of SEQ ID NOS: 08, 09 and 10 in the heavy chain variable domain, and In the light chain variable domain, the HVR of SEQ ID NOS: 12, 05 and 15, or c) in the heavy chain variable domain, the HVR of SEQ ID NOS: 08, 09 and 10, and in the light chain variable domain , HVR of SEQ ID NOS: 13, 14 and 15.

100. The non-covalent complex of any of embodiments 1 to 99, wherein the specific binding The antibody to human Tau (pS422) comprises a) a heavy chain variable domain of SEQ ID NO: 20 and a light chain variable domain of SEQ ID NO: 17, or b) a heavy chain variable domain of SEQ ID NO: 19 and a light chain variable domain of SEQ ID NO: 16, or c) a heavy chain variable domain of SEQ ID NO: 19 and a light chain variable domain of SEQ ID NO: 17, or d) a heavy chain of SEQ ID NO: The variable domain and the light chain variable domain of SEQ ID NO: 17.

The non-covalent complex of any one of embodiments 1 to 100, wherein the non-covalent complex is for treating Alzheimer's disease.

The non-covalent complex of any one of embodiments 1 to 101, wherein the two antibodies in the complex are silencing of effector function.

The non-covalent complex of any one of embodiments 1 to 102, wherein the two antibodies of the complex have no effector function.

The non-covalent complex of any one of embodiments 1 to 103, wherein the antibody i) that specifically binds to human Tau (pS422) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, And/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) fully binds to full length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 01) ID NO: 02), and/or iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422.

The non-covalent complex according to any one of embodiments 1 to 104, wherein the antibody that specifically binds to human Tau (pS422) has the following EC ₅₀ value a) human having the amino acid sequence SEQ ID NO: 03 a Tau (pS422) fragment, 6 ng/mL or less, and/or b) a full length human Tau (pS422) having an amino acid sequence of SEQ ID NO: 02, 4.5 ng/mL or less, and 4.5 ng/mL or less, and / or c) a human Tau (pS422) aggregate having an amino acid sequence of SEQ ID NO: 02, 30 ng/mL or less, and/or d) having an amino acid sequence of SEQ ID NO: 01 and having an amine The human acid Tau of the base acid mutation S422A, 125 ng/mL or less than 125 ng/mL.

106. The non-covalent complex of any of embodiments 1 to 105, wherein specifically binds to human Tau (pS422) (SEQ ID NO: 02) and does not bind to human Tau (SEQ ID NO: 01) antibody.

The non-covalent complex according to any one of embodiments 1 to 106, wherein the antibody that specifically binds to human Tau (pS422) is a monoclonal antibody.

The non-covalent complex according to any one of embodiments 1 to 107, wherein the antibody that specifically binds to human Tau (pS422) is an antibody fragment that binds to human Tau (pS422) and i) specifically binds The polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) specifically binds at position 422 The full length human Tau (SEQ ID NO: 02), and/or iv) phosphorylated at the serine acid, specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422. The aggregate, and/or v) specifically binds to the full length human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) has the amino acid sequence SEQ ID NO: 03 of human Tau (pS422) fragments with 6ng / mL or 6ng / mL or less of EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL or less of EC ₅₀ values, and / or viii) having the amino acid sequence of SEQ ID NO: ₅₀ having a value of 30ng / mL or 30ng / mL of the following EC 02 of human Tau (pS422) aggregates, and / or ix) pairs have Acid sequence of SEQ ID NO: 01 and having the amino acid S422A mutant human Tau values having ₅₀ 125ng / mL or 125ng / mL or less of the EC.

The non-covalent complex according to any one of embodiments 1 to 108, wherein the antibody that specifically binds to human Tau (pS422) is a) a full length antibody of human subclass IgG1, or b) a human subclass of IgG4 Full length antibody, or c) full length antibody to human subclass IgG1 with mutations L234A, L235A and P329G, d) full length antibody to human subclass IgG4 with mutations S228P, L235E and P329G, e) mutations in both heavy chains L234A, L235A and P329G and full-length antibodies of human subclass IgG1 with mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V and Y349C in the other heavy chain, or f) in both heavy chains A full-length antibody of human subclass IgG4 having mutations T228W and P329G and having mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V and Y349C in the other heavy chain.

The non-covalent complex according to any one of embodiments 1 to 109, wherein the antibody a) which specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain comprises the HVR of SEQ ID NO: 08, SEQ ID NO: 18 and SEQ ID NO: 10, ii) the constant region is a human IgG1 constant region, wherein the C-terminal is away from the amino acid residue May exist or absent, and iii) the constant region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) The variable domain comprises the HVR of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, ii) the constant region is the human kappa light chain constant region or the human lambda light chain constant region, and c) i) specific binding The polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) specifically binds at position 422 The full length human Tau (SEQ ID NO: 02), and/or iv) phosphorylated at the serine acid, specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422. Aggregate, / or v) specifically binds to full length human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) to human Tau having the amino acid sequence SEQ ID NO: 03 ( ₅₀ value pS422) fragment has 6ng / mL 6ng / mL or less of the EC, and / or vii) having the amino acid sequence of SEQ ID NO: human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL the following EC ₅₀ values of, and / or viii) having the amino acid sequence of SEQ ID NO: ₅₀ having a value of 30ng / mL or 30ng / mL of the following EC 02 of human Tau (pS422) aggregates, and / or ix) to having the amino acid sequence of SEQ ID NO: 01 and having the human amino acid mutations S422A ₅₀ having a Tau value of 125ng / mL or 125ng / mL or less of the EC.

The non-covalent complex according to any one of embodiments 1 to 110, wherein the antibody a) which specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain comprises the HVR of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10, ii) the constant region is a human IgG1 constant region, wherein the C-terminal amino acid residue May exist or absent, and iii) the constant region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) The variable domain comprises the HVR of SEQ ID NO: 12, SEQ ID NO: 05 and SEQ ID NO: 15, ii) the constant region is the human kappa light chain constant region or the human lambda light chain constant region, and c) i) specific binding The polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) specifically binds at position 422 The full length human Tau (SEQ ID NO: 02), and/or iv) phosphorylated at the serine acid, specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422. Aggregate, / or v) specifically binds to full length human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) to human Tau having the amino acid sequence SEQ ID NO: 03 ( ₅₀ value pS422) fragment has 6ng / mL 6ng / mL or less of the EC, and / or vii) having the amino acid sequence of SEQ ID NO: human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL the following EC ₅₀ values of, and / or viii) having the amino acid sequence of SEQ ID NO: ₅₀ having a value of 30ng / mL or 30ng / mL of the following EC 02 of human Tau (pS422) aggregates, and / or ix) to having the amino acid sequence of SEQ ID NO: 01 and having the human amino acid mutations S422A ₅₀ having a Tau value of 125ng / mL or 125ng / mL or less of the EC.

The non-covalent complex according to any one of embodiments 1 to 111, wherein the antibody a) which specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain comprises the HVR of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10, ii) the constant region is a human IgG1 constant region, wherein the C-terminal amino acid residue May exist or absent, and iii) the constant region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) The variable domain comprises the HVR of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, ii) the constant region is the human kappa light chain constant region or the human lambda light chain constant region, and c) i) specific binding The polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) specifically binds at position 422 The full length human Tau (SEQ ID NO: 02), and/or iv) phosphorylated at the serine acid, specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422. Aggregate, / or v) specifically binds to full length human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) to human Tau having the amino acid sequence SEQ ID NO: 03 ( ₅₀ value pS422) fragment has 6ng / mL 6ng / mL or less of the EC, and / or vii) having the amino acid sequence of SEQ ID NO: human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL the following EC ₅₀ values of, and / or viii) having the amino acid sequence of SEQ ID NO: ₅₀ having a value of 30ng / mL or 30ng / mL of the following EC 02 of human Tau (pS422) aggregates, and / or ix) to having the amino acid sequence of SEQ ID NO: 01 and having the human amino acid mutations S422A ₅₀ having a Tau value of 125ng / mL or 125ng / mL or less of the EC.

The non-covalent complex according to any one of embodiments 1 to 112, wherein the antibody a) which specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 20, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) constant The region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has an amino acid sequence SEQ ID NO :17, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) 1 μg/mL does not bind to full length human Tau (SEQ ID NO: 01), and/or iii) specifically binds to full length human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, And/or iv) aggregates of human Tau (SEQ ID NO: 02) that specifically bind to phosphorylation at serine at position 422, and/or v) specifically bind to an amino acid sequence SEQ ID NO: 01 and full length human Tau with amino acid mutation S422A, and/or vi) EC having 6 ng/mL or 6 ng/mL or less for human Tau (pS422) fragment having amino acid sequence SEQ ID NO: _a value of ₅₀ , and/or vii) having an EC ₅₀ value of 4.5 ng/mL or less or 4.5 ng/mL for a human Tau (pS422) fragment having the amino acid sequence SEQ ID NO: 02, and/or viii) having an amine The human Tau (pS422) aggregate of the SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has an amine group. S422A mutant of human Tau acid having ₅₀ values 125ng / mL or 125ng / mL or less of the EC.

The non-covalent complex according to any one of embodiments 1 to 113, wherein the antibody a) which specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 19, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) constant The region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has an amino acid sequence SEQ ID NO :16, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) 1 μg/mL does not bind to full length human Tau (SEQ ID NO: 01), and/or iii) specifically binds to full length human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, And/or iv) aggregates of human Tau (SEQ ID NO: 02) that specifically bind to phosphorylation at serine at position 422, and/or v) specifically bind to an amino acid sequence SEQ ID NO: 01 and full length human Tau with amino acid mutation S422A, and/or vi) EC having 6 ng/mL or 6 ng/mL or less for human Tau (pS422) fragment having amino acid sequence SEQ ID NO: _a value of ₅₀ , and/or vii) having an EC ₅₀ value of 4.5 ng/mL or less or 4.5 ng/mL for a human Tau (pS422) fragment having the amino acid sequence SEQ ID NO: 02, and/or viii) having an amine The human Tau (pS422) aggregate of the SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has an amine group. S422A mutant of human Tau acid having ₅₀ values 125ng / mL or 125ng / mL or less of the EC.

The non-covalent complex according to any one of embodiments 1 to 114, wherein the antibody a) which specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 19, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) constant The region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has an amino acid sequence SEQ ID NO :17, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) 1 μg/mL does not bind to full length human Tau (SEQ ID NO: 01), and/or iii) specifically binds to full length human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, And/or iv) aggregates of human Tau (SEQ ID NO: 02) that specifically bind to phosphorylation at serine at position 422, and/or v) specifically bind to an amino acid sequence SEQ ID NO: 01 and full length human Tau with amino acid mutation S422A, and/or vi) EC having 6 ng/mL or 6 ng/mL or less for human Tau (pS422) fragment having amino acid sequence SEQ ID NO: _a value of ₅₀ , and/or vii) having an EC ₅₀ value of 4.5 ng/mL or less or 4.5 ng/mL for a human Tau (pS422) fragment having the amino acid sequence SEQ ID NO: 02, and/or viii) having an amine The human Tau (pS422) aggregate of the SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has an amine group. S422A mutant of human Tau acid having ₅₀ values 125ng / mL or 125ng / mL or less of the EC.

The non-covalent complex according to any one of embodiments 1 to 115, wherein the antibody a) which specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 21, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) constant The region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has an amino acid sequence SEQ ID NO :17, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) 1 μg/mL does not bind to full length human Tau (SEQ ID NO: 01), and/or iii) specifically binds to full length human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, And/or iv) aggregates of human Tau (SEQ ID NO: 02) that specifically bind to phosphorylation at serine at position 422, and/or v) specifically bind to an amino acid sequence SEQ ID NO: 01 and full length human Tau with amino acid mutation S422A, and/or vi) EC having 6 ng/mL or 6 ng/mL or less for human Tau (pS422) fragment having amino acid sequence SEQ ID NO: _a value of ₅₀ , and/or vii) having an EC ₅₀ value of 4.5 ng/mL or less or 4.5 ng/mL for a human Tau (pS422) fragment having the amino acid sequence SEQ ID NO: 02, and/or viii) having an amine The human Tau (pS422) aggregate of the SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has an amine group. S422A mutant of human Tau acid having ₅₀ values 125ng / mL or 125ng / mL or less of the EC.

117. The non-covalent complex of any one of embodiments 1 to 116, wherein the antibody that specifically binds to human Tau (pS422) has a proline at positions 4, 24 and 78 in the heavy chain variable domain Residues.

The non-covalent complex of any one of embodiments 1 to 117, wherein the antibody that specifically binds to human Tau (pS422) has a arginine residue at position 71 in the heavy chain variable domain.

119. A haptenated antibody that specifically binds to the blood brain barrier receptor and anti-human A non-covalent complex of Tau (pS422)/hapten bispecific antibody.

120. A non-covalent complex comprising a haptenated antibody and a bispecific antibody that specifically binds to a blood brain barrier receptor, the bispecific antibody having specific binding to a specific binding to a blood brain barrier receptor The first binding specificity and specificity of the hapten of the haptenated antibody binds to the second binding specificity of human Tau (pS422), wherein the haptenized antibody that specifically binds to the blood brain barrier receptor is This second binding specificity of the bispecific antibody specifically binds.

The non-covalent complex of any one of embodiments 119 to 120, wherein the haptenated anti-system that specifically binds to the blood-brain barrier receptor is selected from the group consisting of: specific binding to the blood-brain barrier Biotinylated antibody, a theophylline antibody that specifically binds to the blood-brain barrier receptor, a digoxigenin antibody that specifically binds to the blood-brain barrier receptor, and a carbon boron that specifically binds to the blood-brain barrier receptor Antibody, luciferylated antibody that specifically binds to the blood-brain barrier receptor, a helical antibody that specifically binds to the blood-brain barrier receptor, and a bromodeoxyuridine antibody that specifically binds to the blood-brain barrier receptor .

The non-covalent complex according to any one of embodiments 119 to 121, wherein the blood brain barrier receptor system is selected from the group consisting of: transferrin receptor (TfR), insulin receptor, insulin-like growth factor Receptor (IGF receptor), low density lipoprotein receptor-related protein 8 (LRP8), low density lipoprotein receptor-related protein 1 (LRP1), and heparin-binding epidermal growth factor-like growth factor (HB-EGF).

The non-covalent complex of any one of embodiments 119 to 122, wherein the bispecific antibody is a full length antibody comprising two binding sites.

The non-covalent complex of any one of embodiments 119 to 123, wherein the bispecific antibody is one that has been fused to one or two scFv or scFab or CrossFab or scCrossFab and comprises three or four binding sites Full length antibody.

The non-covalent complex of any one of embodiments 119 to 124, wherein the bispecific anti-system is selected from the group consisting of an antibody fragment, F(ab')2, and a bifunctional antibody.

126. The non-covalent complex of any one of embodiments 119 to 125, wherein the bispecific antibody is a humanized or human antibody.

127. The non-covalent complex of any one of embodiments 119 to 126, wherein the bispecific antibody is free of effector function.

The non-covalent complex of any one of embodiments 119 to 127, wherein the bispecific antibody does not have a functional Fc region in the embodiment.

129. The non-covalent complex of any one of embodiments 119 to 128, wherein the bispecific antibody does not have an Fc region.

The non-covalent complex according to any one of embodiments 119 to 129, wherein the bispecific antibody has an Fc region comprising a human IgG1 subclass of the mutations L234A, L235A and P329G, wherein the positions are according to the Kabat Fc region The number (Kabat EU index) is determined.

The non-covalent complex according to any one of embodiments 119 to 129, wherein the bispecific antibody has an Fc region comprising a human IgG4 subclass of the mutations S228P, L235E and P329G, wherein the positions are according to the Kabat Fc region The number (Kabat EU index) is determined.

The non-covalent complex of any one of embodiments 119 to 131, wherein the bispecific antibody comprises a) a binding site for a hapten that specifically binds to a haptenized antibody of the blood brain barrier receptor Point and a binding site for human Tau (pS422), or b) two binding sites for a hapten that specifically binds to a blood-brain barrier receptor and one for human Tau (pS422) a binding site, or c) a binding site for a hapten that specifically binds to a haptenized antibody of the blood brain barrier receptor and two binding sites for human Tau (pS422), or d) two a binding site for a hapten that specifically binds to a haptenated antibody of the blood brain barrier receptor and two binding sites for human Tau (pS422), wherein in the case of b) and c), the bispecific One of the heavy chains of the antibody comprises a purine mutation and each of the other strands comprises a purine mutation.

133. The non-covalent complex of any one of embodiments 119 to 132, wherein the bispecific antibody comprises two binding sites for a hapten that specifically binds to a haptenized antibody of the blood brain barrier receptor And two binding sites for human Tau (pS422).

134. The non-covalent complex of any one of embodiments 119 to 133, wherein the bispecific antibody specifically binds to a protein 8 specificity associated with (human) transferrin receptor or low density lipoprotein receptor Two binding specificities (anti-hapten binding specificity) of the hapten of the bound haptenated antibody.

135. The non-covalent complex according to any one of embodiments 119 to 134, wherein the binding specificity of digoxin which specifically binds to the digoxigenin antibody which specifically binds to the blood brain barrier receptor is antibody heavy A strand variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 65, (b) comprising a heavy chain CDR2 of the amino acid sequence SEQ ID NO: (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 67, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 69, and (e) comprises the amino acid sequence SEQ ID NO: 70 The light chain CDR2, and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO:71.

136. The non-covalent complex of any one of embodiments 119 to 135, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody that specifically binds to a blood brain barrier receptor is humanized Binding specificity.

137. The non-covalent complex of any one of embodiments 119 to 136, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody that specifically binds to a blood brain barrier receptor comprises, as embodied The CDRs and acceptor human frameworks in Example 17 (e.g., human immunoglobulin framework or human consensus framework).

138. The non-covalent complex according to any one of embodiments 119 to 137, wherein the binding specificity of digoxin which specifically binds to the digoxigenin antibody which specifically binds to the blood brain barrier receptor is antibody heavy A chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 73, (b) comprising an amino acid sequence of SEQ ID NO: 74 The heavy chain CDR2, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 75, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 77, and (e) comprises the amino acid sequence SEQ ID NO: 78 light chain CDR2, and (f) comprises the amino acid sequence SEQ ID NO: 79 light chain CDR3.

139. The non-covalent complex of any one of embodiments 119 to 138, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody that specifically binds to a blood brain barrier receptor is antibody heavy a chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, and the amino acid sequence SEQ ID NO: 68 or 76, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequence.

The non-covalent complex of any one of embodiments 119 to 139, wherein there is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 A %-consistent VH sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-digoxigenin antibody comprising the sequence retains the ability to bind to digoxin. In certain embodiments, a total of 119 to 10 amino acids in SEQ ID NO: 68 or 76 have been substituted, inserted, and/or deleted.

141. The non-covalent complex of any one of embodiments 119 to 140, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

142. The non-covalent complex of any one of embodiments 119 to 141, wherein the digoxigenin binding specificity comprises the VH sequence of SEQ ID NO: 68 or 76, including post-translational modifications of the sequence.

143. The non-covalent complex of any one of embodiments 119 to 142, wherein the binding specificity of digoxin which specifically binds to a digoxigenin antibody that specifically binds to a blood brain barrier receptor is antibody heavy A chain variable domain and an antibody light chain variable domain pair additionally comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with the amino acid sequence SEQ ID NO: 72 or 80 , 97%, 98%, 99% or 100% sequence identity of the light chain variable domain (VL).

144. The non-covalent composite of any one of embodiments 119 to 143, wherein VL sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% less consistent contain substitutions (eg conservative substitutions), insertions or deletions relative to the reference sequence However, the anti-digoxigenin antibody comprising the sequence retains the ability to bind to digoxin. In certain embodiments, a total of 119 to 10 amino acids in SEQ ID NO: 72 or 80 have been substituted, inserted, and/or deleted.

145. The non-covalent complex of any one of embodiments 119 to 144, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

146. The non-covalent complex of any one of embodiments 119 to 145, wherein the digoxigenin binding specificity comprises the VL sequence of SEQ ID NO: 72 or 80, including post-translational modifications of the sequence.

147. The non-covalent complex of any one of embodiments 119 to 134, wherein the bispecific antibody comprises a first binding to biotin that specifically binds to a biotinylated antibody that specifically binds to a blood brain barrier receptor Specificity and specificity bind to the second binding specificity of human Tau (pS422).

148. The non-covalent complex of any one of embodiments 119 to 134 and 147, wherein the bispecific antibody has a specific binding to a biotinylated antibody that specifically binds to a (human) transferrin receptor or The two binding specificities (two avidin binding specificities) of the biotinylated antibody to which the low density lipoprotein receptor-related protein 8 specifically binds and the binding specificity of the specific binding to human Tau (pS422).

149. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 148, wherein the binding specificity of biotin that specifically binds to a biotinylated antibody that specifically binds to a blood brain barrier receptor is An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 81, (b) comprising the amino acid sequence SEQ ID NO: 82 The chain CDR2, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 83, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 85, and (e) comprises the amino acid sequence SEQ ID NO :86 light chain CDR2, and (f) comprising an amino acid sequence SEQ ID NO: 87 light chain CDR3.

The non-covalent complex according to any one of embodiments 119 to 134 and 147 to 149, wherein the binding specificity of the biotin which specifically binds to the biotinylated antibody which specifically binds to the blood brain barrier receptor is Humanization combined with specificity.

151. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 150, wherein the binding specificity of biotin that specifically binds to the biotinylated antibody that specifically binds to the blood brain barrier receptor comprises The CDRs as described in Example 149 and the acceptor human framework (e.g., human immunoglobulin framework or human consensus framework).

152. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 151, wherein the binding specificity of the biotin that specifically binds to the biotinylated antibody that specifically binds to the blood brain barrier receptor is An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 89, (b) comprising an amino acid sequence of SEQ ID NO: 90 The chain CDR2, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 91, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 93, and (e) comprises the amino acid sequence SEQ ID NO The light chain CDR2 of 94, and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO:95.

153. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 152, wherein the binding specificity of biotin that specifically binds to a biotinylated antibody that specifically binds to a blood brain barrier receptor is An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96 with the amino acid sequence SEQ ID NO: 84 or 92 %, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequences.

154. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 153, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% consensus VH sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-biotin antibody comprising the sequence retains binding to biotin Ability.

155. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 154, wherein a total of 119 to 10 amino acids in SEQ ID NO: 84 or 92 have been substituted, inserted and/or deleted.

156. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 155, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

157. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 156, wherein the biotin binding specificity comprises a VH sequence of SEQ ID NO: 84 or 92, including post-translational modifications of the sequence .

158. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 157, wherein the binding specificity of the biotin that specifically binds to the biotinylated antibody that specifically binds to the blood brain barrier receptor is An antibody heavy chain variable domain and an antibody light chain variable domain pair additionally comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence SEQ ID NO: 88 or 96, 96%, 97%, 98%, 99% or 100% sequence identity of the light chain variable domain (VL).

159. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 158, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% identity VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-biotin antibody comprising the sequence retains the ability to bind to biotin.

The non-covalent complex of any one of embodiments 119 to 134 and 147 to 159, wherein a total of 119 to 10 amino acids in SEQ ID NO: 88 or 96 have been substituted, inserted and/or deleted.

161. The non-covalent complex of any one of embodiments 119 to 134 and 147 to 160, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

162. A non-covalent composite according to any one of embodiments 119 to 134 and 147 to 161, Wherein the biotin binding specificity comprises the VL sequence of SEQ ID NO: 88 or 96, including post-translational modifications of the sequence.

163. The non-covalent complex of any one of embodiments 119 to 133, wherein the bispecific antibody comprises a first binding specificity that specifically binds to a theophylline antibody that specifically binds to a blood brain barrier receptor and Specific binding to the second binding specificity of human Tau (pS422).

164. The non-covalent complex of any one of embodiments 119 to 134, wherein the bispecific antibody specifically binds to a theophylline antibody or specifically binds to a (human) transferrin receptor The two binding specificities of the antibody specifically bound by the low density lipoprotein receptor-associated protein 8 (two anti-theophylline binding specificities) and the specific binding specificity to human Tau (pS422).

165. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 164, wherein the binding specificity of the theophylline that specifically binds to the theophylline antibody that specifically binds to the blood brain barrier receptor is resistant a heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 97, (b) a heavy chain comprising the amino acid sequence SEQ ID NO: 98 CDR2, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 99, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 101, and (e) comprises the amino acid sequence SEQ ID NO: The light chain CDR2 of 102 and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO:103.

166. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 165, wherein the binding specificity of the theophylline that specifically binds to the theophylline antibody that specifically binds to the blood brain barrier receptor is Humanization combined with specificity.

167. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 166, wherein the binding specificity of the theophylline that specifically binds to the theophylline antibody that specifically binds to the blood brain barrier receptor comprises The CDRs as described in Example 165 and the acceptor human framework (e.g., human immunoglobulin framework or human consensus framework).

168. A non-covalent composite according to any one of embodiments 119 to 134 and 163 to 167, The binding specificity of theophylline which specifically binds to the theophylline antibody which specifically binds to the blood brain barrier receptor is an antibody heavy chain variable domain and an antibody light chain variable domain pair, which comprises (a) an amino acid. The heavy chain CDR1 of SEQ ID NO: 105, (b) comprises the heavy chain CDR2 of the amino acid sequence SEQ ID NO: 106, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 107, (d) a light chain CDR1 comprising the amino acid sequence SEQ ID NO: 109, (e) comprising the amino acid sequence SEQ ID NO: 110 of the light chain CDR2 and (f) comprising the amino acid sequence SEQ ID NO: 111 of the light chain CDR3 .

169. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 168, wherein the binding specificity of the theophylline that specifically binds to the theophylline antibody that specifically binds to the blood brain barrier receptor is An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96 with the amino acid sequence SEQ ID NO: 100 or 108 %, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequences.

The non-covalent composite of any one of embodiments 119 to 134 and 163 to 169, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% consensus VH sequence contains a substitution (e.g., a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-theaine antibody comprising the sequence retains the ability to bind to theophylline.

171. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 170, wherein a total of 119 to 10 amino acids in SEQ ID NO: 100 or 108 have been substituted, inserted and/or deleted.

172. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 171, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

173. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 172, wherein the theophylline binding specificity comprises the VH sequence of SEQ ID NO: 100 or 108, including post-translational modifications of the sequence.

174. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 173, wherein the binding specificity of theophylline that specifically binds to the theophylline antibody that specifically binds to the blood brain barrier receptor is An antibody heavy chain variable domain and an antibody light chain variable domain pair additionally comprising at least 90%, 91%, 92%, 93%, 94%, 95%, and the amino acid sequence SEQ ID NO: 104 or 112, 96%, 97%, 98%, 99% or 100% sequence identity of the light chain variable domain (VL).

175. The non-covalent composite of any one of embodiments 119 to 134 and 163 to 174, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% identity VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-theaine antibody comprising the sequence retains the ability to bind to theophylline.

176. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 175, wherein a total of 119 to 10 amino acids in SEQ ID NO: 104 or 112 have been substituted, inserted and/or deleted.

177. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 176, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

178. The non-covalent complex of any one of embodiments 119 to 134 and 163 to 177, wherein the theophylline binding specificity comprises the VL sequence of SEQ ID NO: 104 or 112, including post-translational modifications of the sequence.

179. The non-covalent complex of any one of embodiments 119 to 133, wherein the bispecific antibody comprises a first binding specificity that specifically binds to a luciferylated antibody that specifically binds to a blood brain barrier receptor (anti-luciferin binding specificity; anti-FLUO binding specificity) and specific binding specificity to human Tau (pS422).

The non-covalent complex of any one of embodiments 119 to 134 and 179, wherein the bispecific antibody has a specific binding to a luciferylated antibody that specifically binds to a (human) transferrin receptor or An antibody that specifically binds to the low density lipoprotein receptor-associated protein 8 The two binding specificities and specificity bind to the second binding specificity of human Tau (pS422).

181. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 180, wherein the binding specificity of luciferin that specifically binds to a luciferylated antibody that specifically binds to a blood brain barrier receptor An antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 113, (b) comprising an amino acid sequence SEQ ID NO: 114 The heavy chain CDR2, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 115, (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 117, and (e) comprises the amino acid sequence SEQ The light chain CDR2 of ID NO:118 and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO:119.

182. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 181, wherein the binding specificity of luciferin that specifically binds to a luciferylated antibody that specifically binds to a blood brain barrier receptor Sex is the binding specificity of humanization.

183. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 182, wherein the binding specificity of luciferin that specifically binds to a luciferylated antibody that specifically binds to a blood brain barrier receptor Sexually comprises the CDRs as described in Example 63 and the acceptor human framework (e.g., human immunoglobulin framework or human consensus framework).

184. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 183, wherein the binding specificity of luciferin that specifically binds to a luciferylated antibody that specifically binds to a blood brain barrier receptor Is an antibody heavy chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96 with the amino acid sequence SEQ ID NO: 116. %, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequences.

185. The non-covalent composite of any one of embodiments 119 to 134 and 179 to 184, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% consensus VH sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-luciferin antibody comprising the sequence retains binding to luciferin Ability.

186. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 185, wherein a total of 119 to 10 amino acids in SEQ ID NO: 116 have been substituted, inserted and/or deleted.

187. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 186, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

188. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 187, wherein the luciferin binding specificity comprises the VH sequence of SEQ ID NO: 116, including post-translational modifications of the sequence.

189. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 188, wherein the binding specificity of luciferin that specifically binds to a luciferylated antibody that specifically binds to the blood brain barrier receptor Is an antibody heavy chain variable domain and an antibody light chain variable domain pair additionally comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence SEQ ID NO: 96%, 97%, 98%, 99% or 100% sequence identity of the light chain variable domain (VL) sequence.

190. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 189, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% identity VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-luciferin antibody comprising the sequence retains the ability to bind to luciferin.

191. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 190, wherein a total of 119 to 10 amino acids in SEQ ID NO: 120 have been substituted, inserted and/or deleted.

192. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 191, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

193. The non-covalent complex of any one of embodiments 119 to 134 and 179 to 192, Wherein the luciferin binding specificity comprises the VL sequence of SEQ ID NO: 120, including post-translational modifications of the sequence.

194. The non-covalent complex of any one of embodiments 119 to 133, wherein the bispecific antibody comprises a first binding specifically binding to a bromodeoxyuridine antibody that specifically binds to a blood brain barrier receptor Specificity and specificity bind to the second binding specificity of human Tau (pS422).

195. The non-covalent complex of any one of embodiments 119 to 134 and 194, wherein the bispecific antibody specifically binds to bromodeoxyuridine which specifically binds to the (human) transferrin receptor The binding specificity and specificity of the antibody or antibody that specifically binds to the low density lipoprotein receptor-associated protein 8 binds to the second binding specificity of human Tau (pS422).

196. The non-covalent complex of any one of embodiments 119 to 134 and 194 to 195, wherein the bromodeoxyuridine that specifically binds to a bromodeoxyuridine antibody that specifically binds to a blood brain barrier receptor The binding specificity of the glycoside is an antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 121, (b) comprising an amino acid sequence SEQ ID NO: 123 heavy chain CDR2, (c) comprising the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 125, (d) comprising the amino acid sequence SEQ ID NO: 126, the light chain CDR1, (e) comprising an amine The light chain CDR2 of SEQ ID NO: 127 and (f) comprise the light chain CDR3 of the amino acid sequence SEQ ID NO: 128.

197. The non-covalent complex of any one of embodiments 119 to 134 and 194 to 196, wherein the bromodeoxyuridine which specifically binds to a bromodeoxyuridine antibody that specifically binds to a blood brain barrier receptor The binding specificity of the glycoside is humanized binding specificity.

198. The non-covalent complex of any one of embodiments 119 to 134 and 194 to 197, wherein bromodeoxyuridine specifically binds to a bromodeoxyuridine antibody that specifically binds to a blood brain barrier receptor The binding specificity of the glycoside comprises the CDRs as described in the above examples and the acceptor human framework (e.g., human immunoglobulin framework or human common framework).

199. The non-covalent complex of any one of embodiments 119 to 134 and 194 to 198, wherein the bromodeoxyuridine that specifically binds to a bromodeoxyuridine antibody that specifically binds to a blood brain barrier receptor The binding specificity of the glycoside is an antibody heavy chain variable domain and an antibody light chain variable domain pair comprising (a) a heavy chain CDR1 comprising the amino acid sequence SEQ ID NO: 121 or 122, (b) comprising an amino acid The heavy chain CDR2 of SEQ ID NO: 123 or 124, (c) comprises the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 125, and (d) comprises the light chain CDR1 of the amino acid sequence SEQ ID NO: 126, ( e) a light chain CDR2 comprising the amino acid sequence SEQ ID NO: 127 and (f) a light chain CDR3 comprising the amino acid sequence SEQ ID NO: 128.

The non-covalent complex according to any one of embodiments 119 to 134 and 194 to 199, wherein the bromodeoxyuridine which specifically binds to a bromodeoxyuridine antibody which specifically binds to a blood brain barrier receptor The binding specificity of the glycoside is an antibody heavy chain variable domain and an antibody light chain variable domain pair comprising at least 90%, 91%, 92%, 93%, 94 with the amino acid sequence SEQ ID NO: 129 or 131. %, 95%, 96%, 97%, 98%, 99% or 100% sequence identity heavy chain variable domain (VH) sequences.

The non-covalent composite of any one of embodiments 119 to 134 and 194 to 200, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% consensus VH sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-bromodeoxyuridine antibody comprising the sequence retains the ability to bind to bromodeoxyuridine.

The non-covalent complex of any one of embodiments 119 to 134 and 194 to 201, wherein a total of 119 to 10 amino acids in SEQ ID NO: 129 or 131 have been substituted, inserted and/or deleted.

203. The non-covalent complex of any one of embodiments 119 to 134 and 194 to 202, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

The non-covalent complex of any one of embodiments 119 to 134 and 194 to 203, wherein the bromodeoxyuridine binding specificity comprises the VH sequence of SEQ ID NO: 129 or 131, Includes post-translational modifications of the sequence.

205. The non-covalent complex of any one of embodiments 119 to 134 and 194 to 204, wherein bromodeoxyuridine specifically binds to a bromodeoxyuridine antibody that specifically binds to a blood brain barrier receptor The binding specificity of the glycoside is an antibody heavy chain variable domain and an antibody light chain variable domain pair, which additionally comprises at least 90%, 91%, 92%, 93% with the amino acid sequence SEQ ID NO: 130 or 132, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the light chain variable domain (VL) sequence.

The non-covalent composite of any one of embodiments 119 to 134 and 194 to 205, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A 98% or 99% identity VL sequence contains a substitution (eg, a conservative substitution), an insertion or a deletion relative to a reference sequence, but the anti-bromodeoxyuridine antibody comprising the sequence retains the ability to bind to bromodeoxyuridine.

207. The non-covalent complex of any one of embodiments 119 to 134 and 194 to 206, wherein a total of 119 to 10 amino acids in SEQ ID NO: 130 or 132 have been substituted, inserted and/or deleted.

208. The non-covalent complex of any one of embodiments 119 to 134 and 194 to 207, wherein the substitution, insertion or deletion is present in a region outside the CDR (ie, in the FR).

209. The non-covalent complex of any one of embodiments 119 to 134 and 194 to 208, wherein the bromodeoxyuridine binding specificity comprises a VL sequence of SEQ ID NO: 130 or 132, including translation of the sequence Post-modification.

The non-covalent complex of any one of embodiments 119 to 208, wherein the haptenated antibody that specifically binds to the blood brain barrier receptor is in a hapten and an antibody that specifically binds to a blood brain barrier receptor. Contains a linking group.

211. The non-covalent complex of embodiment 210, wherein the linking group is a peptide linking group.

212. The non-covalent complex of embodiment 210, wherein the linking group is a chemical linker Group (non-peptide linking group).

213. The non-covalent complex of any one of embodiments 119 to 212, wherein the antibody that specifically binds to the blood brain barrier receptor is a full length antibody.

214. The non-covalent complex of any one of embodiments 119 to 213, wherein the antibody i) that specifically binds to human Tau (pS422) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, And/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) fully binds to full length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 01) ID NO: 02), and/or iv) an aggregate that specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422, and/or v) specifically binds to Amino acid sequence SEQ ID NO: 01 and human Tau with amino acid mutation S422A.

215. The non-covalent complex of any one of embodiments 119 to 214, wherein the antibody that specifically binds to human Tau (pS422) comprises a) in the heavy chain variable domain, SEQ ID NOs: 08, 18 and HVR of 10, or b) HVR of SEQ ID NOS: 08, 09 and 10 in the heavy chain variable domain.

216. The non-covalent complex of any one of embodiments 119 to 215, wherein the antibody that specifically binds to human Tau (pS422) additionally comprises a) in the light chain variable domain, SEQ ID NO: 13, 14 And HVR of 15 or b) HVR of SEQ ID NOS: 12, 05 and 15 in the light chain variable domain.

217. The non-covalent complex of any one of embodiments 119 to 216, wherein the antibody that specifically binds to human Tau (pS422) comprises a) in the heavy chain variable domain, SEQ ID NOs: 08, 18 and HVR of 10, and HVR of SEQ ID NOS: 13, 14 and 15 in the light chain variable domain, or b) HVR of SEQ ID NOS: 08, 09 and 10 in the heavy chain variable domain, and Light chain In the variable domain, HVR of SEQ ID NOS: 12, 05 and 15, or c) in the heavy chain variable domain, HVR of SEQ ID NOS: 08, 09 and 10, and in the light chain variable domain, SEQ ID NO: HVR of 13, 14 and 15.

218. The non-covalent complex of any one of embodiments 119 to 217, wherein the antibody that specifically binds to human Tau (pS422) comprises a) the heavy chain variable domain of SEQ ID NO: 20 and SEQ ID NO: a light chain variable domain of 17 or b) a heavy chain variable domain of SEQ ID NO: 19 and a light chain variable domain of SEQ ID NO: 16, or c) a heavy chain variable domain of SEQ ID NO: 19 and The light chain variable domain of SEQ ID NO: 17, or d) the heavy chain variable domain of SEQ ID NO: 21 and the light chain variable domain of SEQ ID NO: 17.

219. The non-covalent complex of any one of embodiments 119 to 218, wherein the non-covalent complex is for treating Alzheimer's disease.

The non-covalent complex of any one of embodiments 119 to 219, wherein the two antibodies in the complex are silencing of effector function.

221. The non-covalent complex of any one of embodiments 119 to 220, wherein the two antibodies of the complex have no effector function.

222. The non-covalent complex of any one of embodiments 119 to 221, wherein the antibody i) that specifically binds to human Tau (pS422) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, And/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) fully binds to full length human Tau phosphorylated at serine at position 422 (SEQ ID NO: 01) ID NO: 02), and/or iv) specifically binds to aggregates of human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422.

Human 03: 223. The non-covalent complex of Example 119 according to the embodiment 222, wherein the specific binding to human Tau (pS422) the antibody has the following EC ₅₀ values of a) having amino acid sequence SEQ ID NO a Tau (pS422) fragment, 6 ng/mL or less, and/or b) a full length human Tau (pS422) having an amino acid sequence of SEQ ID NO: 02, 4.5 ng/mL or less, and 4.5 ng/mL or less, and / or c) a human Tau (pS422) aggregate having an amino acid sequence of SEQ ID NO: 02, 30 ng/mL or less, and/or d) having an amino acid sequence of SEQ ID NO: 01 and having an amine The human acid Tau of the base acid mutation S422A, 125 ng/mL or less than 125 ng/mL.

224. The non-covalent complex of any one of embodiments 119 to 223, wherein specifically binds to human Tau (pS422) (SEQ ID NO: 02) and does not bind to human Tau (SEQ ID NO: 01) antibody.

225. The non-covalent complex of any one of embodiments 119 to 224, wherein the antibody that specifically binds to the blood brain barrier receptor is a monoclonal antibody.

226. The non-covalent complex of any one of embodiments 119 to 225, wherein the antibody that specifically binds to human Tau (pS422) is an antibody fragment that binds to human Tau (pS422) and i) specifically binds The polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) specifically binds at position 422 The full length human Tau (SEQ ID NO: 02), and/or iv) phosphorylated at the serine acid, specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422. The aggregate, and/or v) specifically binds to the full length human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) has the amino acid sequence SEQ ID NO: 03 of human Tau (pS422) fragments with 6ng / mL or 6ng / mL or less of EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL or less of EC ₅₀ values, and / or viii) having the amino acid sequence of SEQ ID NO: ₅₀ having a value of 30ng / mL or 30ng / mL of the following EC 02 of human Tau (pS422) aggregates, and / or ix) pair Amino acid sequence SEQ ID NO: 01 and having the amino acid S422A mutant human Tau values having ₅₀ 125ng / mL or 125ng / mL or less of the EC.

227. The non-covalent complex of any one of embodiments 119 to 226, wherein the antibody that specifically binds to the blood brain barrier receptor is a) a full length antibody to human subclass IgG1, or b) a human subclass IgG4 Full length antibody, or c) full length antibody to human subclass IgG1 with mutations L234A, L235A and P329G, d) full length antibody to human subclass IgG4 with mutations S228P, L235E and P329G, e) mutations in both heavy chains L234A, L235A and P329G and full-length antibodies of human subclass IgG1 with mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V and Y349C in the other heavy chain, or f) in both heavy chains Mutant S228P, L235E and P329G and have full-length antibodies to human heavy-chain T366W and S354C in one heavy chain and human subclass IgG4 with mutations T366S, L368A, Y407V and Y349C in another heavy chain, or g) in two heavy chains Full length antibody to human subclass IgG1 with mutations L234A, L235A and P329G and having mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V and Y349C in the other heavy chain, or h) in two heavy chains Has mutations S228P, L235E and P329G and Heavy chain A full length antibody of human subclass IgG4 having the mutations T366W and Y349C and having the mutations T366S, L368A, Y407V and S354C in the other heavy chain.

228. The non-covalent complex of any one of embodiments 119 to 227, wherein antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain comprises the HVR of SEQ ID NO: 08, SEQ ID NO: 18 and SEQ ID NO: 10, ii) the constant region is a human IgG1 constant region, wherein the C-terminal is away from the amino acid residue May exist or absent, and iii) the constant region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) The variable domain comprises the HVR of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, ii) the constant region is the human kappa light chain constant region or the human lambda light chain constant region, and c) i) specific binding The polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) specifically binds at position 422 The full length human Tau (SEQ ID NO: 02), and/or iv) phosphorylated at the serine acid, specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422. Aggregate, And/or v) specifically binds to full length human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) to human Tau having the amino acid sequence SEQ ID NO: (pS422) fragments with 6ng / mL or 6ng / mL or less of EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: the human Tau 02 (pS422) fragments with 4.5ng / mL or 4.5 ng / the EC ₅₀ values mL or less, and / or viii) having the amino acid sequence of SEQ ID NO: ₅₀ having a value of 30ng / mL or 30ng / mL or less of the EC 02 of human Tau (pS422) aggregates, and / or ix) The human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A has an EC ₅₀ value of 125 ng/mL or less.

229. The non-covalent complex of any one of embodiments 119 to 228, wherein antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain comprises the HVR of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10, ii) the constant region is a human IgG1 constant region, wherein the C-terminal amino acid residue May exist or absent, and iii) the constant region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) The variable domain comprises the HVR of SEQ ID NO: 12, SEQ ID NO: 05 and SEQ ID NO: 15, ii) the constant region is the human kappa light chain constant region or the human lambda light chain constant region, and c) i) specific binding The polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) specifically binds at position 422 The full length human Tau (SEQ ID NO: 02), and/or iv) phosphorylated at the serine acid, specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422. Aggregate, And/or v) specifically binds to full length human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) to human Tau having the amino acid sequence SEQ ID NO: (pS422) fragments with 6ng / mL or 6ng / mL or less of EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: the human Tau 02 (pS422) fragments with 4.5ng / mL or 4.5 ng / the EC ₅₀ values mL or less, and / or viii) having the amino acid sequence of SEQ ID NO: ₅₀ having a value of 30ng / mL or 30ng / mL or less of the EC 02 of human Tau (pS422) aggregates, and / or ix) The human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A has an EC ₅₀ value of 125 ng/mL or less.

The non-covalent complex according to any one of embodiments 119 to 229, wherein the antibody a) which specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain comprises the HVR of SEQ ID NO: 08, SEQ ID NO: 09 and SEQ ID NO: 10, ii) the constant region is a human IgG1 constant region, wherein the C-terminal amino acid residue May exist or absent, and iii) the constant region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) The variable domain comprises the HVR of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, ii) the constant region is the human kappa light chain constant region or the human lambda light chain constant region, and c) i) specific binding The polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) specifically binds at position 422 The full length human Tau (SEQ ID NO: 02), and/or iv) phosphorylated at the serine acid, specifically binds to human Tau (SEQ ID NO: 02) phosphorylated at the serine at position 422. Aggregate And/or v) specifically binds to full length human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A, and/or vi) to human Tau having the amino acid sequence SEQ ID NO: (pS422) fragments with 6ng / mL or 6ng / mL or less of EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: the human Tau 02 (pS422) fragments with 4.5ng / mL or 4.5 ng / the EC ₅₀ values mL or less, and / or viii) having the amino acid sequence of SEQ ID NO: ₅₀ having a value of 30ng / mL or 30ng / mL or less of the EC 02 of human Tau (pS422) aggregates, and / or ix) The human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A has an EC ₅₀ value of 125 ng/mL or less.

231. The non-covalent complex of any one of embodiments 119 to 230, wherein the antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 20, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) constant The region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has an amino acid sequence SEQ ID NO :17, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) 1 μg/mL does not bind to full length human Tau (SEQ ID NO: 01), and/or iii) specifically binds to full length human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, And/or iv) aggregates of human Tau (SEQ ID NO: 02) that specifically bind to phosphorylation at serine at position 422, and/or v) specifically bind to an amino acid sequence S EQ ID NO: 01 and full length human Tau with amino acid mutation S422A, and/or vi) 6 ng/mL or 6 ng/mL or less for human Tau (pS422) fragment having amino acid sequence SEQ ID NO: EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: having human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL or less of EC ₅₀ values, and / or viii) to The human Tau (pS422) aggregate of the amino acid sequence SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has an amine. The human Tau of the base acid mutation S422A has an EC ₅₀ value of 125 ng/mL or less.

232. The non-covalent complex of any one of embodiments 119 to 231, wherein the antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 19, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) constant The region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has an amino acid sequence SEQ ID NO :16, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) 1 μg/mL does not bind to full length human Tau (SEQ ID NO: 01), and/or iii) specifically binds to full length human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, And/or iv) aggregates of human Tau (SEQ ID NO: 02) that specifically bind to phosphorylation at serine at position 422, and/or v) specifically bind to an amino acid sequence SE QID NO: 01 and full length human Tau with amino acid mutation S422A, and/or vi) 6 ng/mL or 6 ng/mL or less for human Tau (pS422) fragment having amino acid sequence SEQ ID NO: EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: having human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL or less of EC ₅₀ values, and / or viii) to The human Tau (pS422) aggregate of the amino acid sequence SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has an amine. The human Tau of the base acid mutation S422A has an EC ₅₀ value of 125 ng/mL or less.

233. The non-covalent complex of any one of embodiments 119 to 232, wherein antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 19, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) constant The region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has an amino acid sequence SEQ ID NO :17, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) 1 μg/mL does not bind to full length human Tau (SEQ ID NO: 01), and/or iii) specifically binds to full length human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, And/or iv) aggregates of human Tau (SEQ ID NO: 02) that specifically bind to phosphorylation at serine at position 422, and/or v) specifically bind to an amino acid sequence SE QID NO: 01 and full length human Tau with amino acid mutation S422A, and/or vi) 6 ng/mL or 6 ng/mL or less for human Tau (pS422) fragment having amino acid sequence SEQ ID NO: EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: having human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL or less of EC ₅₀ values, and / or viii) to The human Tau (pS422) aggregate of the amino acid sequence SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has an amine. The human Tau of the base acid mutation S422A has an EC ₅₀ value of 125 ng/mL or less.

234. The non-covalent complex of any one of embodiments 119 to 233, wherein antibody a) that specifically binds to human Tau (pS422) comprises two antibody heavy chains, each heavy chain comprising a heavy chain variable domain and a heavy chain constant region, wherein i) the variable domain has the amino acid sequence SEQ ID NO: 21, ii) the constant region is a human IgG1 constant region, wherein the C-terminal can be present or absent from the amino acid residue, and iii) constant The region comprises amino acid changes L234A, L235A and P329G, b) comprises two antibody light chains, each light chain comprising a light chain variable domain and a light chain constant domain, wherein i) the variable domain has an amino acid sequence SEQ ID NO :17, ii) the constant region is a human kappa light chain constant region or a human lambda light chain constant region, and c) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) 1 μg/mL does not bind to full length human Tau (SEQ ID NO: 01), and/or iii) specifically binds to full length human Tau (SEQ ID NO: 02) phosphorylated at serine at position 422, And/or iv) aggregates of human Tau (SEQ ID NO: 02) that specifically bind to phosphorylation at serine at position 422, and/or v) specifically bind to an amino acid sequence SE QID NO: 01 and full length human Tau with amino acid mutation S422A, and/or vi) 6 ng/mL or 6 ng/mL or less for human Tau (pS422) fragment having amino acid sequence SEQ ID NO: EC ₅₀ values, and / or vii) having the amino acid sequence of SEQ ID NO: having human Tau (pS422) with fragment 02 of 4.5ng / mL or 4.5ng / mL or less of EC ₅₀ values, and / or viii) to The human Tau (pS422) aggregate of the amino acid sequence SEQ ID NO: 02 has an EC ₅₀ value of 30 ng/mL or less, and/or ix) has an amino acid sequence of SEQ ID NO: 01 and has an amine. The human Tau of the base acid mutation S422A has an EC ₅₀ value of 125 ng/mL or less.

235. The non-covalent complex of any one of embodiments 119 to 234, wherein the antibody that specifically binds to human Tau (pS422) has a proline at positions 4, 24 and 78 in the heavy chain variable domain Residues.

236. The non-covalent complex of any one of embodiments 119 to 235, wherein the antibody that specifically binds to human Tau (pS422) has a arginine residue at position 71 in the heavy chain variable domain.

237. A pharmaceutical formulation comprising the non-covalent complex of any of embodiments 1 to 236 and a pharmaceutically acceptable carrier.

238. The pharmaceutical formulation of embodiment 237, wherein the pharmaceutical formulation additionally comprises an additional therapeutic agent.

239. The pharmaceutical formulation of embodiment 238, wherein the additional therapeutic agent is an anti-amyloid therapeutic agent.

240. The pharmaceutical formulation of embodiment 239, wherein the anti-amyloid therapeutic agent is an anti-human alpha-synuclein antibody or an anti-Aβ antibody.

241. The pharmaceutical formulation of any one of embodiments 239 to 122, wherein the anti-human alpha-synuclein antibody or the anti-Abeta antibody is haptenized.

242. The pharmaceutical formulation of any one of embodiments 239 to 123, wherein the anti-human alpha-synuclein antibody or anti-Abeta antibody is complexed with an anti-blood brain barrier receptor/hapten bispecific antibody.

243. A non-covalent complex according to any one of embodiments 1 to 236 for use as a medicament.

244. A non-covalent complex according to any one of embodiments 1 to 236 for use in the treatment of Alzheimer's disease.

245. A non-covalent complex according to any one of embodiments 1 to 236, which is used before treatment Driven Alzheimer's disease.

246. The non-covalent complex of any of embodiments 1 to 236 for use in the treatment of mild Alzheimer's disease.

247. The non-covalent complex of any of embodiments 1 to 236 for use in ameliorating Tau (pS422)-induced neurodegeneration.

248. The non-covalent complex of any of embodiments 1 to 236 for use in maintaining cognition and function.

249. The non-covalent complex of any one of embodiments 1 to 236 for use in slowing the rate of cognitive and dysfunction.

250. Use of a non-covalent complex according to any one of embodiments 1 to 236 for the manufacture of a medicament.

251. The use of embodiment 250, wherein the medicament is for treating Alzheimer's disease.

252. The use of any one of embodiments 250 to 251, wherein the medicament is for treating prodromal Alzheimer's disease.

253. The use of any one of embodiments 250 to 251, wherein the medicament is for the treatment of mild Alzheimer's disease.

254. The use of any one of embodiments 250 to 251, wherein the agent is for alleviating Tau (pS422)-induced neurodegeneration.

255. The use of any one of embodiments 250 to 251, wherein the medicament is for maintaining cognition and function.

256. The use of any one of embodiments 250 to 251, wherein the medicament is for slowing the rate of cognitive and dysfunction.

257. A method of treating an individual having Alzheimer's disease, comprising administering to the individual an effective amount of a non-covalent complex of any of embodiments 1 to 236.

258. A method of alleviating Tau (pS422)-induced neurodegeneration in an individual comprising administering to the individual an effective amount of a non-covalent complex as in any one of embodiments 1 to 236 to reduce Light Tau (pS422)-induced neurodegeneration.

259. A method of maintaining the cognition and function of an individual comprising administering to the individual an effective amount of a non-covalent complex of any of embodiments 1 to 236 to maintain cognition and function.

260. A method of slowing the rate of cognitive and dysfunction in an individual comprising administering to the individual an effective amount of a non-covalent complex of any of embodiments 1 to 236 to slow the rate of cognitive and dysfunction.

261. Use of a non-covalent complex according to any one of embodiments 1 to 236 for attenuating Tau (pS422) induced neurodegeneration.

262. Use of a non-covalent complex according to any one of embodiments 1 to 236 for maintaining cognition and function.

263. Use of a non-covalent complex according to any one of embodiments 1 to 236 for slowing cognitive and dysfunctional rates.

264. Use of a non-covalent complex according to any one of embodiments 1 to 236 for the treatment of Alzheimer's disease.

265. Use of a non-covalent complex according to any one of embodiments 1 to 236 for protection against Alzheimer's disease.

266. Use of a non-covalent complex according to any one of embodiments 1 to 236 for stopping the progression of Alzheimer's disease.

267. Use of a non-covalent complex according to any one of embodiments 1 to 236 for preventing the spread of human Tau (pS422)-associated Alzheimer's disease.

268. Use of a non-covalent complex according to any one of embodiments 1 to 236 for reducing lysosomal membrane disintegration.

269. Use of a non-covalent complex according to any one of embodiments 1 to 236 for stabilizing and/or disintegrating a stable lysosomal membrane induced by human Tau (pS422).

270. Use of a non-covalent complex according to any one of embodiments 1 to 236 for preventing progression of Alzheimer's disease.

V. Example

The following are examples of the methods and compositions of the present invention. It will be appreciated that various other embodiments may be practiced in the light of the general description provided above.

Materials and methods Recombinant DNA technology

DNA was manipulated using standard methods as described in Sambrook, J. et al, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Use molecular biological reagents according to the manufacturer's instructions.

Gene and oligonucleotide synthesis

The desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthetic gene fragment was cloned into E. coli plastids for propagation/amplification. The DNA sequence of the subcloned gene fragment was examined by DNA sequencing. Alternatively, short synthetic DNA fragments can be assembled by adhesion to chemically synthesized oligonucleotides or via PCR. Individual oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

Reagent

If not otherwise specified, all commercially available chemicals, antibodies, and kits are used as provided by the manufacturer's protocol.

Example 1 Preparation and purification of rabbit antibodies Immunology

NZW rabbits from Charles River Laboratories International, Inc. were used for immunization. The phosphopeptide Tau (416-430) [pS422] coupled to KLH was dissolved in K ₃ PO ₄ buffer (pH 7.0) at a concentration of 1 mg/ml and mixed with complete Freund's adjuvant (CFA). (1:1) until a stable emulsion is produced. Three rabbits received an intradermal (id) injection of 2 ml of the emulsion at intervals of one week, followed by a second intramuscular (im) and a third subcutaneous (sc) injection of 1 ml each. The fourth 1 ml intramuscular injection was performed two weeks later, followed by two additional 1 ml subcutaneous injections at four week intervals. 10 ml of each animal peripheral blood sample was collected 4 to 6 days after the third, fourth, fifth and sixth injections and used for single cell sorting in FACS. An additional 0.5 ml of each animal serum was also collected and used to determine the Tau (416-463) [pS422] specific antibody response.

Antibody response

The antibody response to immunization was determined by serial dilution of serum using an ELISA in an ELISA pre-coated streptavidin 96-well microtiter plate (MC1347, Micro Coat Biotechnologie GmbH, Bernried, Germany). 30 ng of each well of biotinylated Tau (416-430) [pS422] was incubated overnight in 1 x PBS at 4 °C. For detection, goat anti-rabbit IgG (The Jackson laboratory) conjugated with horseradish peroxidase at a dilution of 1:16,000 was used. BM Blue POD from Roche Diagnostics GmbH was subjected to mass spectrometry, precipitated tetramethylbenzidine (TMB), and a stock solution for observation. The reaction was stopped with 1 N HCl and was taken on a Tecan Infinite by 450/690 nm.

B cell colonization Coating plate

Mixture of 3 biotinylated control peptides in PBS (unphosphorylated Tau (416-430), MCAK_human (88-102) [95-pSer] and MAP2_human (1802) at room temperature -1816) [pSer-1802]) or biotinylated phosphopeptide Tau (416-430) [pS422] (each at a concentration of 0.5 μg/ml to 1 μg/ml) to culture sterile streptavidin coated 6 wells The culture plate (cell culture grade) was continued for 1 hour. The plates were washed three times in sterile PBS prior to use. Cell culture was coated with 2 μg/ml KLH (key hole limpet haemocyanine) in carbonate buffer (0.1 M sodium bicarbonate, 34 mM disodium hydrogencarbonate, pH 9.55) at 4 °C. The 6-well culture plate was overnight. The plates were washed three times in sterile PBS prior to use.

Isolation of rabbit peripheral blood mononuclear cells (PBMC)

Rabbit PBMCs were isolated by diluting twice the whole blood containing EDTA with 1 x PBS prior to density centrifugation of mammalian lymphocyte separation fluid (Cedarlane Laboratories). PBMC were washed twice before staining with antibodies.

EL-4 B5 medium

Supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2 mM glutamic acid, 1% penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05 mM β-mercaptoethanol (Gibco, Paisley, Scotland) RPMI 1640 (Pan Biotech, Aidenbach, Germany)

Consumption of macrophages/monocytes

Macrophages and monocytes were consumed by non-specific adhesion using a sterile 6-well culture plate (cell culture grade). The wells were coated with KLH (keyhole spirulina) or streptavidin and a control peptide. Each well is filled with 4ml of Medium and up to at most ⁶ × 10 6 were from immunization of rabbits and the peripheral blood mononuclear cells at 37 [deg.] C allowed the binding in the incubator for 1 hour. 50% of the cells in the supernatant were used for the panning step; the remaining 50% of the cells were directly subjected to immunofluorescence staining and single cell sorting.

Panning B cells on peptides

A 6-well tissue culture plate coated with streptavidin and biotinylated peptide Tau (416-430) [pS422] was inoculated with up to 6 × 10 ⁶ cells per 4 ml of medium and allowed to incubate at 37 ° C in an incubator Combine for 1 hour. Unadhered cells were removed by carefully washing the wells 1-2 times with 1 x PBS. The remaining viscous cells were separated by trypsin for 10 minutes at 37 ° C in an incubator and then washed twice in the medium. The cells were kept on ice until immunofluorescence staining.

Immunofluorescence staining and single cell sorting

Anti-rabbit IgG FITC for single cell sorting was from AbD Serotec (STAR121F, Düsseldorf, Germany). For surface staining, cells from the depletion and panning steps were incubated with PBS containing anti-rabbit IgG FITC antibody for 30 minutes at 4 ° C in the dark Medium, rolling in the cold room. After centrifugation, the supernatant was removed by aspiration. PBMCs were subjected to 2 cycles of centrifugation and washed with ice cold PBS. Finally, PBMC were resuspended in ice cold PBS and immediately subjected to FACS analysis. Propidium iodide (BD Pharmingen, San Diego, CA, USA) at a concentration of 5 μg/ml was added prior to FACS analysis to identify dead cells and viable cells. FACS was performed using a Becton Dickinson FACSAria equipped with FACSDiva software (BD Biosciences, USA) and a single FITC-labeled live cell was deposited in a 96-well culture dish.

B cell culture

B cell cultures were prepared by methods similar to those described by Zubler, RH et al., J. Immunol. 134 (1985) 3662-3668. Briefly, Pansorbin cells (1:20000) (Calbiochem (Merck), Darmstadt, Deutschland), 5% rabbit thymocyte supernatant and gamma radiation EL-4-B5 mouse thymoma cells (2 x 10 ^{4 )} /well) 210 μl/well EL-4 B5 medium Single-class B cells were cultured in a 96-well culture dish at 37 ° C in a 5% CO ₂ atmosphere for 7 days in an incubator. B cell culture supernatants were removed for screening and cells were harvested immediately for variable region gene selection or frozen at -80 °C in 100 μl RLT buffer (Qiagen, Hilden, Germany).

B cell pure line screening

B cell culture supernatants bound to biotinylated Tau (416-430) [pS422] were screened by ELISA. Non-phosphorylated Tau (416-430), KLH (keyhole spirulina) and the unrelated phosphopeptide MCAK_human (88-102) [95-pSer] were used as control antigens. To prepare an ELISA plate, a streptavidin-precoated microtiter plate was incubated with 50 ng/ml biotinylated Tau (415-430) [pS422] for 1 hour at room temperature. KLH or control peptide was coated at 1 μg/ml. B cell supernatants were diluted 1:5 to 1:10 and incubated for 60 minutes in antigen coated microtiter plates. After extensive washing, the binding of rabbit antibodies was detected using goat anti-rabbit IgG digoxin binding to a detection antibody (Chemicon AQ301D). After incubation with TMB at room temperature, the absorbance at 370 nm to 492 nm was measured. Enter one Step by considering a B cell line with biotinylated Tau (416-430) [pS422] but without KLH and MCAK_human (88-102) [95-pSer] producing the above background signal and performing variable region gene selection .

PCR amplification and sequencing of V domain

Using NucleoSpin ^® 8/96 RNA kit (Macherey &Nagel; 740709.4,740698) was prepared according to the manufacturer programs all RNA. All steps were performed on an epMotion 5075 liquid operating system (Eppendorf). The RNA was detached with 60 μl of RNase free water. Superscript III First-Strand Synthesis SuperMix (Invitrogen; 18080-400) and oligo dT-primer were used according to the manufacturer's instructions to generate cDNA by reverse transcriptase reaction using 6 μl of RNA. 4 μl of cDNA was used to use the primers rbHCfinal.up and rbHCfinal.do for the heavy chain with AccuPrime SuperMix (Invitrogen; 12344-040) in a final volume of 50 μl, and rbLCfinal.up and rbLCfinal.do for the light chain (see table below), To amplify immunoglobulin heavy and light chain variable regions (VH and VL). The PCR conditions were as follows: hot start at 94 ° C for 5 minutes; 20 seconds at 94 ° C, 20 seconds at 70 ° C, 35 cycles at 68 ° C for 45 seconds, and finally extended at 68 ° C for 7 minutes.

8 μl of 50 μl of the PCR solution was loaded on 48 E-Gel 2% (Invitrogen G8008-02). Using NucleoSpin ^® Extract II kit according to the manufacturer programs (Macherey &Nagel; 740609250) cleaning and positive PCR reaction in a 50μl buffer in the eluting fractions thereof. CtrlHCfinal.up and rbHCfinal.do were used for the heavy chain and 12 μl of the purified PCR product was directly sequenced in both directions using rbLCfinal.up and rbLCfinal.do (see table above) for the light chain.

Recombinant performance of rabbit monoclonal antibody and rabbit/mouse chimeric antibody

Recombinant expression of rabbit monoclonal antibodies by drape selection method (Haun, RS et al, BioTechniques 13 (1992) 515-518; Li, MZ, et al, Nature Methods 4 (2007) 251-256) The PCR product encoding VH or VL is cloned into the expression vector as cDNA. The linearized expression plastid encoding the VL of the rabbit κ or γ constant region and the VH insert was amplified by PCR using overlapping primers. The purified PCR product was incubated with T4 DNA-polymerase to produce a single-strand suspension. The reaction was stopped by the addition of dCTP. In the next step, the plastid and insert are combined and incubated with RecA, which induces site-specific recombination. The recombinant plasmid was transformed into E. coli. On the next day, growth colonies were selected by plastid preparation, restriction analysis, and DNA-sequencing and appropriate recombinant plasmids were tested. Regarding antibody expression, the isolated HC and LC plastids were co-transfected into HEK293 cells and the supernatant was collected 1 week later. Regarding the selection and expression of chimeric antibodies in rabbit mice, the VH and VL regions were amplified by PCR and subcultured in expression vectors containing mouse constant kappa or mouse constant gamma 1 region. The rabbit/mouse chimeric HC and LC plastids were tested for appropriate insertion isolation by restriction analysis and DNA sequencing and transiently co-transfected into HEK293 cells. The supernatant was collected one week after transfection.

Antibody purification

On MabSelectSuRe ^TM column (GE Healthcare), the cell culture supernatant from recombinant expression of rabbit antibody purification. The column was equilibrated with 25 mmol/L Tris-HCl, 25 mmol/L NaCl (pH 7.4) before loading the sample. Isolation of the antibody was achieved with 50 mmol/L acetate (pH 3.14). The lysed sample was immediately loaded onto a desalting column (Sephadex G25, GE Healthcare) and dissolved in 20 mmol/L His-HCl, 140 mmol/L NaCl (pH 6.0). This buffer is also used to store purified antibodies. Typical storage temperatures are 4 ° C, room temperature during the purification process and -80 ° C after aliquots. On MabSelectSuRe ^TM column (GE Healthcare), was purified from rabbit cell culture supernatant of the recombinant expression / mouse chimera antibodies. The column was equilibrated with 1 x PBS (pH 7.4) before loading the sample. Isolation of the antibody was achieved with 100 mmol/L citrate (pH 3.0). The dissolved sample was immediately neutralized with 2 mol/L Tris/HCl (pH 9.0). Subsequently, the antibody was loaded on a size exclusion column (Superdex 200, GE Healthcare), and eluted in 20 mmol/L His-HCl, 140 mmol/L NaCl (pH 6.0). This buffer is also used to store purified antibodies. Typical storage temperatures are 4 ° C, room temperature during the purification process and -80 ° C after aliquots.

Example 2 Anti-Tau pS422 single rabbit antibody is highly selective for Tau phosphorylated at pS422 and binds to fiber aggregates of Tau pS422 ELISA

Recombinant rabbit monoclonal antibodies were expressed in HEK 293 cells. Binding to biotinylated Tau (416-430) [pS422], non-phosphorylated Tau (416-430), KLH (keyhole spirulina) and unrelated phosphopeptide MCAK_human by ELISA (88- 102) [95-pSer] test cell culture supernatant or purified rabbit antibody. To prepare an ELISA plate, a streptavidin-precoated microtiter plate was incubated with 50 ng/ml biotinylated Tau (415-430) [pS422] for 1 hour at room temperature. KLH or control peptide was coated at 1 μg/ml. Rabbit anti-Tau pS422 antibody (Abeam AB51071) or rabbit antibody containing supernatant was incubated for 60 minutes at various concentrations in antigen-labeled microtiter plates. After extensive washing, the binding of rabbit antibodies was detected using goat anti-rabbit IgG digoxin binding to a detection antibody (Chemicon AQ301D). After incubation with TMB at room temperature, the absorbance at 370 nm to 492 nm was measured. Antibody binding is characterized by its EC50 value. Antibodies that bind to biotinylated Tau (416-430) [pS422] and non-phosphorylated Tau (416-430) peptides are characterized by their EC50 values. Cross-reactivity with KLH or MCAK phosphopeptides was estimated by single point measurements at higher concentrations, i.e., 1:5 dilutions of cell culture supernatants. The results are shown in the table below. The EC50 value of binding to the Tau phosphopeptide was found to be 100-fold lower than the EC50 value bound to the Tau peptide, indicating that the selectivity of the phosphorylated Tau fragment is at least higher than that of the non-phosphorylated Tau peptide. 100 times. At the background level all antibodies bind to KLH and MCAK control phosphopeptides, which are about 1 < 3% of the maximum measured value of Tau phosphopeptide.

The specificity of soluble and aggregated full length Tau pS422 was also tested. Fiber aggregates of Tau pS422 (300 μg/ml) were coated onto a polystyrene-based Maxisorb microtiter plate (Nunc) overnight at room temperature. Soluble full length Tau and Tau pS422 were coated onto a Maxisorb microtiter plate in a similar manner. Rabbit anti-Tau pS422 antibody control (Abeam AB51071) or purified rabbit antibody was added and incubated for 60 minutes at a concentration of up to 1000 ng/ml. After extensive washing, the binding of rabbit antibodies was detected using goat anti-rabbit IgG digoxin binding to a detection antibody (Chemicon AQ301D). After incubation with TMB at room temperature, the absorbance at 370 nm to 492 nm was measured. Antibody binding is characterized by its EC50 value. The results are shown in the table below.

The rabbit monoclonal antibody binds to the Tau-pS422 protein with an EC50 value of less than 1 ng/ml. The EC50 value of the fiber Tau pS422 was detected in the range of 0.4 ng/ml to 14 ng/ml. Signals that bind to the non-phosphorylated full length Tau protein cannot be distinguished from background levels. Therefore, according to It is estimated that each of the antibodies binds to Tau pS422 and fiber Tau pS422 with a selectivity of at least 100-fold compared to Tau.

BIAcore ^TM

Further study and analysis by BIAcore ^TM confirm binding of fiber aggregate and Tau pS422. Measured at 37 ° C using a BIAcore 3000 instrument. The system and sample buffer were HBS-EP (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% polysorbate 20 (v/v)). BIAcore ^TM CM5 sensor chip of the pretreatment procedure. 0.1% SDS, 50 mM NaOH, 10 mM HCl, and 100 mM H ₃ PO _{4 were} sequentially injected through the flow cells FC1, FC2, FC3, and FC4 for 30 seconds. According to the manufacturer instructions using a BIAcore 3000 ^TM v.4.1 guide the amine coupling procedure. After activation of the EDC/NHS on the surface of the sensor, the non-phosphate selective anti-Tau antibody mAb <TAU>M-4/53-IgG was immobilized on the sensor channels FC2, FC3 and FC4. An antibody recognizing an unrelated antigen was captured on FC1 against CK-MM (creatine kinase isotype) as a control. The antibody capture system was fixed at 30 μg/ml by diluting mAb<TAU>M-4/53-IgG and antibody against CK-MM in 10 mM NaAc (pH 5.0) and injecting at 10 μl/min for 7 minutes contact time to fix 10.000 RU. . The surface was deactivated by saturation with 1 M ethanolamine. The conditions of the sensor were as follows: 5 μl/min of phosphorylated filamentous Tau protein (0.3 mg/ml of a stock diluted 1:100 in HBS-EP) was used as a dissolved analyte for 2 min. Regeneration was carried out with 10 mM glycine (pH 2.5) at 30 μl/min for 3 minutes. It was speculated that the analyte bound to mAb 4/53 did not dissociate pTau fibrils because no dissociation of pTau filaments from mAb 4/53 was observed. For all other measurement cycles, 0.3 mg/ml pTau fibrils were diluted 1:100 in HBS-EP buffer and injected at 10 μl/min for 1 min to present pTau to individual antibody analytes in a heterogeneous sandwich mode. Antibody analytes were diluted in HBS-EP buffer to a concentration of 100 nM and injected into the system at 20 μl/min for 3 minutes. After 3 minutes of dissociation, the sensor surface was regenerated by injecting 10 mM glycine (pH 2.5) twice at 100 μl/min for 1 minute followed by washing with 100 μl/min HBS for 15 seconds. Monitor the combination and dissociation phases of interactions. Since the antibody analyte in solution state is bivalent, the affinity-affinitive antibody pTau kinetics is characterized by a two-phase dissociation model that consists of a rapid early dissociation step based on affinity followed by affinity in the dissociation of the latter complex. Stable but limited speed kinetic steps. At the end of the analyte injection 10 seconds (front) and 50 seconds (post), kd and t/2 (dissociation) were quantified where possible. The kinetic measurements were evaluated using a dual reference program. First, the signal from the FC1 reference is subtracted to correct for buffer overall effects and non-specific binding. Second, the 0 nM analyte injection was subtracted to correct for dissociation of the primary antibody from the respective capture system. The BIAcore ^TM evaluation software v.4.1, using Langmuir fitting model evaluation 1.1 dissociation kinetic rate. The antigen/antibody complex stability half-life (min) was calculated according to the formula ln(2)/60*kd.

The results are summarized in the table below.

Example 3 Binding of anti-Tau pS422 monoclonal antibody to intracellular pTau in brain sections of patients with Alzheimer's disease

The specificity and sensitivity of pTau lesions in the brain tissue of Alzheimer's disease by a single rabbit anti-Tau pS422 antibody was studied by immunofluorescence staining experiments using cryosections of human brain tissue from AD patients. Sexual immunohistochemistry detection. This procedure was essentially the same as described in Example X (mouse antibody). Binding by secondary antibody (Invitrogen / Molecular Probes A11034) of goat anti-rabbit Alexa Fluor488 ^® detect rabbit IgG. For pure Mab 005, Mab 019, Mab 020, Mab 085, Mab 086 and Mab 097, the specificity and sensitivity staining of pTau deposits and fibrils is evident. Intracellular pTau deposition such as larger neurofibrillary tangles and elongated neutrophil thin lines is evident. The minimum effective concentration range for all pure lines studied was determined to be between 0.08 μg/ml and 0.016 μg/ml, indicating a high sensitivity binding to true human pTau deposits.

Example 4 Humanization of rabbit anti-human Tau (pS422) antibody

As used herein, a "variable domain" (variable domain of a light chain (VL), a variable domain of a heavy chain (VH)) indicates each of a pair of light and heavy chain domains that are directly involved in antibodies and Binding of Tau (pS422) antigen. The variable light and heavy chain domains have the same general structure and each domain comprises four framework regions that are widely conserved, linked by three "hypervariable regions".

The structure of the VH and VL domains of rabbit antibody monoclonal antibody 086 was analyzed by computer and compared with the structural database of human VH and VL domains (IMGT). The most similar V domain map was selected to graft the CDRs of rabbit antibodies to selected human VH and VL domains. In addition, the similarity of the primary sequence is considered by comparing the VH and VL domain first-order sequences of the rabbit antibody with the human V-domain antibody library to narrow the selection range of the human V domain. In some humanized variants, a back mutation in the human framework region is introduced into the rabbit parental residue. Similarly, mutations in the CDRs are introduced into the variants as appropriate to potentially increase antigen affinity, thereby maintaining the CDR tertiary structure and thereby removing undesirable features, such as cysteine residues or after antibody purification. The modified residue is made.

The heavy and light chain vectors containing each of the humanized variants were co-transfected into HEK293 suspension cells in a microtiter culture dish to obtain the full complement of all possible light chain/heavy chain combinations. Cell culture of size IgG. After 5 days of incubation at 37 ° C, the supernatant was collected and purified by protein A affinity chromatography on a microtiter scale.

Example 5 Recombinant expression vector a) production of a vector expressing an immunoglobulin heavy chain using a human IgG1 constant region

The human IgG1 constant region (CH1, hinge, CH2, CH3) and home-derived are assembled by fusing a DNA fragment encoding a VH domain of a specific anti-human Tau (pS422)-specific antibody to a sequence element encoding a human IgG1 constant region. The humanized heavy chain encoding the fusion gene of the humanized anti-human Tau (pS422) antibody VH domain of the rabbit antibody Mab 086.

The human IgG1 constant region has the following amino acid sequence: (SEQ ID NO: 58).

The expression vector also contains an origin of replication from the vector pUC18, which allows for replication of this plastid in E. coli, and the beta-endosinase gene, which confers ampicillin resistance in E. coli.

The transcriptional unit of the antibody heavy chain contains the following functional elements in the 5' to 3' direction: - immediate early fortifier and promoter of human cytomegalovirus (P-CMV) including intron A, - human heavy chain immunoglobulin Protein 5'-untranslated region (5'UTR), - murine immunoglobulin heavy chain signal sequence, - heavy chain variable (VH) domain encoding nucleic acid, - nucleic acid encoding human IgG1 constant region, and - bovine growth hormone Polyadenylation sequence (BGH pA).

b) production of a vector expressing an immunoglobulin light chain using a human Ig-kappa constant region

Human Ig-kappa constant region (CL-κ) and its origin are assembled by fusing a DNA fragment encoding a respective anti-human Tau (pS422) antibody VL (κ) domain to a sequence element encoding a human Ig-kappa constant region. The humanized kappa light chain encoding the fusion gene of the rabbit antibody Mab 086 against the human Tau (pS422) antibody VL (κ) domain.

The human Ig-kappa constant region has the following amino acid sequence: (SEQ ID NO: 59).

The expression vector also contains an origin of replication from the vector pUC18, which allows for replication of this plastid in E. coli, and the beta-endosinase gene, which confers ampicillin resistance in E. coli.

The transcriptional unit of the antibody kappa light chain contains the following functional elements in the 5' to 3' direction: - immediate early fortifier and promoter of human cytomegalovirus (P-CMV) including intron A, - human heavy chain immunization Globulin 5'-untranslated region (5'UTR), - murine immunoglobulin heavy chain signal sequence, - light chain variable (VL) domain encoding nucleic acid, - nucleic acid encoding human Ig-kappa constant region, and - Bovine growth hormone polyadenylation sequence (BGH pA).

c) generating a vector expressing an immunoglobulin light chain using a human Ig-λ constant region

The human Ig-λ constant region (CL-λ) and its origin are assembled by fusing a DNA fragment encoding the respective anti-human Tau (pS422) antibody VL (λ) domain to a sequence element encoding a human Ig-λ constant region. The humanized lambda light chain of the rabbit antibody Mab 086 against the human Tau (pS422) antibody VL (λ) domain encodes a fusion gene.

The human Ig-λ constant region has the following amino acid sequence: (SEQ ID NO: 60).

The expression vector also contains an origin of replication from the vector pUC18, which allows for replication of this plastid in E. coli, and the beta-endosinase gene, which confers ampicillin resistance in E. coli.

The transcriptional unit of the antibody lambda light chain contains the following functional elements in the 5' to 3' direction: - an immediate early enhancer and promoter of human cytomegalovirus (P-CMV) including intron A, - human heavy chain immunization a 5'-untranslated region (5'UTR), a murine immunoglobulin heavy chain signal sequence, a nucleic acid encoding a light chain variable (VL) domain, a nucleic acid encoding a human Ig-λ constant region, and - Bovine growth hormone polyadenylation sequence (BGH pA).

d) production of a vector expressing an immunoglobulin kappa light chain using a human Ig-kappa constant region

Human Ig-kappa constant region (CL-κ) and its origin are assembled by fusing a DNA fragment encoding a respective anti-human Tau (pS422) antibody VL (κ) domain to a sequence element encoding a human Ig-kappa constant region. The human Ig-kappa light chain encoding the fusion gene of the rabbit antibody Mab 086 against the human Tau (S422) antibody VL (κ) domain. The construct is in the genomic tissue, that is, the intron exists in the signal peptide and between the VL (κ) and CL-κ domains.

The expression vector also contains an origin of replication from the vector pUC18, which allows for replication of this plastid in E. coli, and the beta-endosinase gene, which confers ampicillin resistance in E. coli.

The transcriptional unit of the antibody kappa light chain contains the following functional elements in the 5' to 3' direction: - immediate early fortifier and promoter of human cytomegalovirus (P-CMV), - human heavy chain immunoglobulin 5'- Translation area (5'UTR), - murine immunoglobulin heavy chain signal sequence, - light chain variable (VL) domain encoding nucleic acid, - human IgG κ constant region, and - bovine growth hormone polyadenylation sequence (BGH pA).

e) generating a vector expressing the immunoglobulin lambda light chain using a human Ig-λ constant region

The coding assembly comprises a human Ig-λ constant region (CL-λ) and a source by fusing a DNA fragment encoding a respective anti-human Tau (pS422) antibody VL (λ) domain to a sequence element encoding a human Ig-λ constant region. The human Ig-λ light chain of the rabbit antibody Mab 086 against the human Tau (S422) antibody VL (λ) domain encodes a fusion gene. The construct is in the genomic tissue, ie the intron is present in the signal peptide and between the VL (λ) and CL-λ domains.

The expression vector also contains an origin of replication from the vector pUC18, which allows for replication of this plastid in E. coli, and the beta-endosinase gene, which confers ampicillin resistance in E. coli.

The transcriptional unit of the antibody lambda light chain contains the following functional elements in the 5' to 3' direction: - immediate early fortifier and promoter of human cytomegalovirus (P-CMV), - human heavy chain immunoglobulin 5'- Translational region (5'UTR), - murine immunoglobulin heavy chain signal sequence, - light chain variable (VL) domain encoding nucleic acid, - human IgG lambda constant region, and - bovine growth hormone polyadenylation sequence (BGH) pA).

Example 6 Recombinant production of anti-human Tau (pS422) antibody

Antibodies were generated in transiently transfected HEK293 cells (derived from human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). For transfection of the respective vectors as described in Example 5, a "293"-free transfection reagent (Novagen) was used. Antibodies are expressed from individual plastids. Transfection as specified in the manufacturer's instructions. Transfection from three days to seven days Thereafter, the cell culture supernatant containing the recombinant antibody was collected. The supernatant was stored at low temperature (eg -80 °C) until purification.

General information regarding the recombinant expression of human immunoglobulins in, for example, HEK293 cells is provided in the following literature. Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Example 7 Purification of recombinant anti-human Tau (pS422) antibody

The culture supernatant containing the antibody was filtered and purified by two chromatography steps.

The antibody was captured by affinity chromatography using HiTrap MabSelect SuRe (GE Healthcare) equilibrated with PBS (1 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 137 mM NaCl, 2.7 mM KCl) (pH 7.4). The unbound protein was removed by washing with equilibration buffer, and the antibody was recovered with 25 mM citrate buffer (pH 3.1), which was followed by adjustment of the eluate to pH 6.0 with 1 M Tris base (pH 9.0).

On the size exclusion Superdex 200 ^TM (GE Healthcare) is used as a second chromatographic purification step. Size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The antibody-containing solution was concentrated and stored at -80 °C using an Ultrafree-CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, MA, USA).

Example 8 Kinetic screening

Kinetic screening was performed on a BIAcore 4000 instrument equipped with a BIAcore CM5 sensor according to Schraeml et al. (Schraeml, M. and M. Biehl, Methods Mol. Biol. 901 (2012) 171-181). The BIAcore 4000 appliance is controlled by the V1.1 software. The BIAcore CM5 tandem S-wafer was mounted in an appliance and hydrodynamically processed and pre-treated according to the manufacturer's instructions. The device buffer was HBS-EP buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w/v) P20). Antibody capture system Prepared on the surface of the sensor. Multiple goat anti-human antibodies with human IgG-Fc specificity (Jackson Lab.) were fixed to the device in 10 mM sodium acetate buffer (pH 5) at 30 μg/ml using NHS/EDC chemistry at 10,000 RU. At positions 1, 2, 4 and 5 of the flow cells 1, 2, 3 and 4. The antibody was captured at position 1 and position 5 in each flow cell. Position 2 and position 4 are used as reference positions. The sensor was deactivated with a 1 M ethanolamine solution. A humanized antibody derivative at a concentration between 44 nM and 70 nM was applied to an appliance buffer supplemented with 1 mg/ml CMD (carboxymethyl dextran). The antibody was injected at a flow rate of 30 μl/min for 2 minutes. The surface exhibits the capture level (CL) of the antibody as measured in rel. reaction units (RU). The analyte in the form of a solution, phosphorylated human tau protein, non-phosphorylated human tau protein, and phosphorylated human tau mutein T422S were injected at a flow rate of 300 nM, 30 μl/min for 3 minutes. Monitor dissociation for 5 minutes. The capture system was regenerated by injecting 10 mM glycine buffer (pH 1.7) through all chutes at 30 μL/min for 1 minute. At the two reporting points, the recorded signal shortly before the end of the analyte injection is indicated as late binding (BL); and the recorded signal shortly before the end of the dissociation time, expressed as late stabilization (SL), which is used to characterize the kinetic screening performance. Furthermore, the dissociation rate constant kd (1/s) was calculated according to the Langmuir model and the antibody/antigen complex half-life was calculated in minutes according to the formula ln(2)/(60*kd). The molar ratio (MR) is calculated according to the formula MR = (late binding (RU)) / (capture level (RU)) * (MW (antibody) / (MW (antigen)). The sensor is configured to have a suitable amount In the case of antibody ligand capture levels, each antibody should be functionally capable of binding to at least one analyte in the form of a solution, expressed as a molar ratio MR = 1.0. Next, the molar ratio is also the price of the analyte binding. An indication of the number mode. For antibodies that bind two analytes, the maximum valence can be MR = 2, each with a Fab valence.

In another embodiment, the same experimental equipment was used at 25 ° C and 37 ° C but various analytes in various concentrations were used in solution (at 0 nM (buffer), 1.2 nM, 3.7 nM, 11.1 nM, The kinetic rate was determined at 33.3 nM, 100 nM and 300 nM. According to the manufacturer's instructions and the Langmuir 1.1 model with overall RMAX, Concentration-dependent binding status The software calculation kinetic data was evaluated using BIAcore.

Example 9 ELISA

Adding non-biotinylated peptides/proteins/aggregates to uncoated Maxisorb plates and adding biotinylated peptide/protein/aggregate containing PBS to the streptavidin coated Maxisorb plates And cultivate overnight. The supernatant was discarded and the wells were washed three times with 90 μl of wash buffer (1 x PBS / 0.1% Tween 20). Residual reactive spots were blocked by blocking buffer (1 x PBS/2% BSA (bovine serum albumin dissolving V, no fatty acid, Roche, catalog number: 10735078001) / 0.05% Tween 20) by incubation for one hour. The supernatant was discarded and the wells were washed three times with 90 μl of wash buffer. Samples and control antibodies (1×PBS/0.5% BSA (bovine serum albumin-dissolved V, no fatty acids,) were prepared in ELISA buffer at a starting concentration of 500 ng/mL in 12 dilutions (1:2). Roche, catalog number: 10735078001) / 0.05% Tween 20). The incubation time was 60 minutes on the shaker at room temperature. The supernatant was discarded and the wells were washed three times with 90 μl of wash buffer. A secondary antibody solution was prepared in an ELISA buffer. A total of 25 μl of the antibody mixture was transferred to all wells of the assay plate and thereafter the plate was incubated on a shaker for 60 minutes at room temperature. The supernatant was discarded and the wells were washed three times with 90 μl of wash buffer. 25 μl of ABTS solution was added to all wells. The absorbance was read at 405 nm to 492 nm.

Example 10 Combination of recombinant humanized avidin antibody with biotinylated compound (haptenized compound)

In order to determine whether the humanization procedure and subsequent introduction of a cysteine mutation resulted in a derivative that retained complete binding activity, the following experiment was performed.

The binding properties of the recombinant avidin antibody derivative were analyzed by the Biofilm Interference (BLI) technique using an Octet QK instrument (Fortebio Inc.). This system is well established for studying molecular interactions. The BLi technique is based on the measurement and internal reference of the interference pattern of white light reflected from the surface of the top of the biosensor. The combination of molecules and biosensor tips This results in a measurable interference pattern shift. To analyze whether the humanization procedure described above reduces the ability of avidin antibodies to bind to biotin, the properties of the ability of chimeric and humanized antibodies to bind to biotinylated proteins are directly compared. Binding studies were performed by capturing anti-biotin antibodies on an anti-huIgG Fc antibody capture (AHC) biosensor (Fortebio Inc.). First, the biosensor was incubated for 1 minute in a solution of antibody at a concentration of 0.5 mg/ml in 20 mM histidine, 140 mM NaCl (pH 6.0). Thereafter, the biosensor was incubated for 1 minute in 1 x PBS pH 7.4 to reach a stable baseline. Binding was measured by incubating the antibody-coated biosensor for 5 minutes in a solution containing biotinylated protein at a concentration of 0.06 mg/ml in 20 mM histidine, 140 mM NaCl (pH 6.0). Dissociation was monitored for 5 minutes in 1 x PBS pH 7.4. The resulting binding curves of chimeric and humanized avidin antibodies were directly compared.

The humanized version of the antibody shows equal or even better biotinylated antigen binding compared to the chimeric antibody. Biotinylated proteins show reduced non-specific binding to biosensor residues when the biosensor is coated with Herceptin, which does not bind biotin. Thus, the functional group of the anti-biotin antibody (defined by the sequences as depicted in SEQ ID NOs: 92 and 96) is retained in its humanized variant.

Surface plasmon resonance

Surface plasmon resonance measurements were performed on a BIAcore® T200 instrument (GE Healthcare Biosciences AB, Sweden) at 25 °C. A 4300 Resonance Unit (RU) capture system on a CM3 wafer (GE Healthcare, BR-1005-36) at pH 5.0 using a standard amine coupling kit (BR-1000-50) supplied by GE Healthcare (10 μg/ml anti-human capture (IgG Fc) from Human Antibody Capture Kit, BR-1008-39, GE Healthcare Biosciences AB, Sweden). The working buffer for amine coupling was HBS-N (10 mM HEPES, pH 7.4, 150 mM NaCl, GE Healthcare, BR-1006-70). The procedure used for subsequent binding studies and the dilution buffer was PBS-T (10 mM phosphate buffered saline, including 0.05% Tween 20) pH 7.4. The humanized avidin antibody was captured by injecting a 2 nM solution at a flow rate of 5 μl/min for 60 seconds. Biotinylated siRNA (1:3 serial dilution) was diluted with PBS-T at a concentration of 0.14-100 nM. Binding was measured by injecting each concentration for 180 seconds at a flow rate of 30 μl/min with a dissociation time of 600 seconds. The surface was regenerated by washing with a 3 M MgCl ₂ solution for 30 seconds at a flow rate of 5 μl/min. Data were assessed using the BIA Evaluation Software (GE Healthcare Biosciences AB, Sweden). The overall refractive index difference was corrected by subtracting the reaction obtained from the surface of the anti-human IgG Fc. Blank injection (= secondary reference) is also subtracted. To calculate the KD and kinetic parameters, a Langmuir 1:1 model was used.

Kinetic binding assays were performed on humanized avidin antibodies SEQ ID NO: 92 and 96 by surface plasmon resonance (SPR). An anti-biotin antibody at a concentration of 2 nM was captured by an anti-human IgG Fc antibody conjugated to a CM3 sensor wafer. Binding of biotinylated siRNA (Mw: 13868 Da) at concentrations of 0.41, 1.23, 3.7, 11.1, 33.3, 100 and 300 nM was recorded. Measured in duplicate. Humanized anti-biotin antibody was calculated K _D 0.633nM.

Example 11 Producing a non-covalent complex of a haptenated compound and an anti-hapten antibody General method:

The production of a complex of an anti-hapten antibody and a haptenated compound (= hapten in combination with a payload) will result in a defined complex and should ensure that the compound (= payload) retains its activity in such complexes . In order to produce the hapten-hapten antibody with the respective anti-compounds complexes, hapten compounds were dissolved in H ₂ O to a final concentration of 1mg / ml. The antibody was concentrated to a final concentration of 1 mg/ml (4.85 μM) in 20 mM histidine buffer, 140 mM NaCl, pH=6.0. The haptenated payload and antibody were mixed by aspiration up and down to a 2:1 molar ratio (compound to antibody) and incubated for 15 minutes at room temperature.

Alternatively, the haptenated compound is dissolved in 100% DMF to a final concentration of 10 mg/ml. Concentrate the antibody to 50 mM Tris-HCl, 1 mM EDTA, pH=8.2 The final concentration was 10 mg/ml. The haptenated compound was mixed with the antibody by up-and-down aspiration to a 2.5:1 molar ratio (compound to antibody) and incubated for 60 minutes at room temperature and 350 rpm.

Exemplary method for forming a complex of a haptenated polypeptide and an anti-hapten antibody-digoxigenin-PYY(3-36)/anti-digoxigenin antibody complex

To generate a non-covalent complex of the digoxigenin polypeptide and the anti-digoxigenin antibody, the antibody produced by the murine fusion tumor (freeze from 10 mM KPO ₄ , 70 mM NaCl; pH 7.5) was dissolved in 12 ml of water and was for 20 mM. A solution of histidine, 140 mM NaCl, pH 6.0 dialysis gave 300 mg (2 x 10 ^-6 mol) of 11 ml buffer (c = 27.3 mg/ml). 2.85 mg of digoxin-PYY(3-36) conjugate (11.57 mg, 4 x 10 ^-6 mol, 2 equivalents) was added in 4 portions over 1 hour and incubated for an additional hour at room temperature. After completion of the complex reaction, the complex of 20 mM histidine, 140 mM NaCl, pH 6.0 was purified by size exclusion chromatography at a flow rate of 2.5 ml/min over a Superdex 200 26/60 GL column (320 ml). The dissociated complex was collected into 4 ml of the fraction, concentrated and sterilized through a 0.2 μm filter to obtain 234 mg of a complex having a concentration of 14.3 mg/ml. In a similar manner, in order to produce humanized anti-digoxin antibody complexes, antibody in 20mM histidine, 140mM NaCl, pH 6.0 adjusted to a concentration of 10.6mg / ml of (9.81mg, 0.93ml of 6.5 × 10 ^{- 8} mol). To the antibody solution was added 0.57 mg = 1.97 x 10 ^-7 mol = 3.03 equivalent of digoxigenin polypeptide (DIG-PYY) in the form of a lyophilizate. The polypeptide and antibody were incubated for 1.5 hours at room temperature. Excess polypeptide in 20 mM histidine, 140 mM NaCl, pH 6.0 was removed by size exclusion chromatography on a Superose 6 10/300 GL column at a flow rate of 0.5 ml/min. The lysed complex was collected into 0.5 ml of the fraction, concentrated and sterilized by a 0.2 μm filter to obtain 4.7 mg of a complex having a concentration of 1.86 mg/ml.

The resulting haptenated polypeptide-anti-hapten antibody complex is defined as a monomeric IgG-like molecule that exhibits a single peak in size exclusion chromatography. The resulting complex is defined as a monomeric IgG-like molecule, each carrying two digoxin-PYY derivatives. The defined composition of these peptide complexes was confirmed by size exclusion chromatography, which also indicates the absence of protein aggregates. By SEC- MALS (size exclusion chromatography - multi-angle light scattering) further confirmed the defined composition (and 2:1 polypeptide to protein ratio) of these bispecific peptide complexes. For SEC-MALS analysis, 100-500 μg of each sample was applied to a Superdex 20010/300 GL size exclusion column at a flow rate of 0.25-0.5 ml/min with 1 x PBS pH 7.4 as the mobile phase. Light scattering was detected with a Wyatt MiniDawn TREOS/QELS detector and the refractive index was measured with a Wyatt Optilab rEX-detector. The data obtained were analyzed using software ASTRA (version 5.3.4.14). The results of the SEC-MALLS analysis provide information on the mass, radius and size of the composite. This data is then compared to the data for the corresponding uncomplexed antibody. The results of these experiments indicate that digoxin-PYY exposure to anti-digoxigenin antibodies produces a complex of two digoxin-PYY derivatives per antibody molecule. Thus, digoxigenin PYY can be complexed with an anti-digoxigenin antibody at a defined site (binding region) and in a defined stoichiometry.

Additional evidence for the characterization of complexes by surface plasmon resonance studies provides a complex reaction to produce a defined and fully complexed molecule. Anti-digoxigenin antibodies can bind to the SPR wafer, which results in increased signal. Subsequent addition of the digoxin-PYY conjugate resulted in further signal increase until it completely occupied the entire binding site. Under these conditions, adding more digoxin-PYY does not increase the signal further. This indicates that the complex reaction is specific and the signal is not caused by non-specific adhesion of the digoxigenin polypeptide.

Exemplary method for forming a complex of a haptenated polypeptide and an anti-hapten antibody-Ac-PYY-PEG3-Cys-PEG2-Biot)/chimeric anti-biotin antibody complex

To generate a non-covalent complex of a biotinylated-PYY-polypeptide containing a cysteine linking group, 0.16 mg of Ac-PYY-PEG3-Cys-PEG2-Biot was dissolved in 100% DMF to 10 mg/ml. concentration. An antibody having a concentration of 10.7 mg/ml (about 73 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, and pH 8.2 was used. Ac-PYY-PEG3-Cys-PEG2-Biot was mixed with antibody at a molar ratio of 2.5:1 (Ac-PYY-PEG3-Cys-PEG2-Biot specific antibody) and incubated for 60 minutes at room temperature and 350 rpm. After size exclusion chromatography, the resulting complex was defined as 63% monomeric IgG-like molecule and 37% dimerized soluble aggregate body. The resulting complex was further analyzed by SDS-PAGE followed by Western blot analysis. 10 μg of the complex was mixed with 4×LDS sample buffer (Invitrogen) and incubated at 95 ° C for 5 minutes. Samples were applied to 4-12% Bis-Tris polyacrylamide gel (NuPAGE, Invitrogen) and operated at 200 V and 120 mA for 35 minutes. The molecules separated in the polypropylene guanamine-gel were transferred to a PVDF membrane (0.2 μm pore size, Invitrogen) at 25 V and 160 mA for 40 minutes. The membrane was blocked in 1% (w/v) skim milk in 1 x PBST (1 x PBS + 0.1% Tween 20) for 1 hour at room temperature. Membranes were washed 3X in IX PBST for 5 minutes and subsequently incubated with streptavidin-POD-conjugate (2900 U/ml, Roche), which was used at 1:2000 dilution. Detection of biotin-bound streptavidin-POD on the membrane was performed using Lumi-Light Western dot substrate (Roche).

Exemplary method for forming a complex of a haptenated polypeptide and an anti-hapten antibody-Ac-PYY(PEG3-Cys-PEG2-5-Fluo)/chimeric anti-luciferin antibody complex

To generate a non-covalent complex of a luciferin-binding-PYY-polypeptide containing a cysteine linking group, 0.33 mg of Ac-PYY (PEG3-Cys-PEG2-5-Fluo was dissolved in 100% DMF to 10 mg) Concentration of /ml. Using a concentration of 9.99 mg/ml (about 68 μM) in a buffer consisting of 50 mM Tris-HCl, 1 mM EDTA, pH 8.2, with a molar ratio of 2.5:1 (Ac-PYY (PEG3- Cys-PEG2-5-Fluo) was mixed with Ac-PYY (PEG3-Cys-PEG2-5-Fluo and antibody, and incubated for 60 minutes at room temperature and 350 rpm. Size exclusion chromatography, the resulting complex Defined as 76% monomeric IgG-like molecule and 24% dimeric soluble aggregate. The resulting complex was further analyzed by SDS-PAGE and subsequently detected for luciferin-related fluorescence in polypropylene guanamine-gel. 8 μg complex The material was mixed with 4 x LDS sample buffer (Invitrogen) and incubated for 5 minutes at 95° C. Fluorescence-related fluorescence was recorded using an LumiImager F1 apparatus (Roche) at an excitation wavelength of 645 nm.

Example 12 Polypeptide retention and binding to anti-hapten antibodies

Polypeptides that have previously been shown to be part of a non-covalent hapten-polypeptide conjugate and complexed with an anti-hapten antibody retain functionality (WO 2011/003557, WO 2011/003780 and PCT/EP2011/074273).

Example 13 Engineering of blood-brain barrier-swing module

The hapten-binding bispecific blood-brain barrier-shuttle module is produced by the fusion of a disulfide-stabilized hapten-binding single-chain Fv with the C-terminus of the CH3 domain of an anti-TfR antibody. Applications and techniques similar to those previously described are applied (see for example WO 2014/006124). An example of the composition of such blood-brain barrier-swirl modules is shown in FIG.

The blood-brain barrier-shuttle module recognizes a cell surface target (blood-brain barrier receptor) capable of transcytosis on endothelial cells of the blood-brain barrier. For example, we use two different antibodies that bind to the transferrin receptor with different affinities. The antibody TfR1 binds to the transferrin receptor with high affinity and the antibody TfR2 binds to the transferrin receptor with reduced affinity (see for example WO 2012/075037). The TfR binding site derived from such anti-TfR antibodies is set to enter the unmodified Fab arm of the bispecific antibody to obtain a bivalent full length IgG module. The disulfide-stabilized hapten-binding single-chain Fv is fused to the C-terminus of the CH3 domain of the bispecific antibody produced by a short GS linking group. For example, as the above-described anti-hapten binding site, an entity that binds to a derivative of digo or biotin (Bio) is used (see above for the sequence).

Examples of sequence composition of such shuttle vectors are listed as SEQ ID NO: 134 (LC anti-TfR1 antibody), SEQ ID NO: 135 (HC anti-TfR1 antibody conjugated to scFv anti-digoxigenin antibody fragment), SEQ ID NO: 136 (HC anti-TfR1 antibody conjugated to scFv avidin antibody fragment), SEQ ID NO: 137 (LC anti-TfR2 antibody), SEQ ID NO: 138 (HC anti-binding to scFv anti-digoxigenin antibody fragment) TfR2 antibody), SEQ ID NO: 139 (HC anti-TfR2 antibody conjugated to scFv anti-antibiotic antibody fragment).

Example 14 Performance and purification of bispecific antibodies (blood brain barrier-shuttle module)

The blood-brain barrier-shuttle module bispecific antibody is produced in mammalian cells in a defined serum-free medium (see above) as previously described. HEK293 suspension cells were transiently transfected with L-chain and H-chain encoding plastids to produce cultures that exhibited a blood-brain barrier-shuttle module bispecific antibody.

To produce a digoxigenin payload with high affinity binding to TfR in combination with a blood-brain barrier-shuttle module comprising a plastid/expression cassette of the nucleic acid encoding SEQ ID NO: 134 and a nucleic acid encoding the gene encoding SEQ ID NO: 135 Performance plastid/performance cardinal co-transfection.

To produce a digoxigenin payload with high affinity binding to TfR in combination with a blood-brain barrier-shuttle module comprising a plastid/expression cassette of the nucleic acid encoding SEQ ID NO: 134 and a nucleic acid encoding the gene encoding SEQ ID NO: 136 Performance plastid/performance cardinal co-transfection.

To produce a digoxigenin payload combined with a reduced affinity for binding to TfR in combination with a blood-brain barrier-shuttle module comprising a nucleic acid encoding a nucleic acid of SEQ ID NO: 137 and a nucleic acid encoding the gene encoding SEQ ID NO: 138 The performance of the plastid/performance card was co-transfected.

In order to generate a digoxigenin payload combined with a reduced affinity for binding to TfR in combination with a blood-brain barrier-swirl module, the plastid/expression cassette containing the nucleic acid encoding SEQ ID NO: 137 and the nucleic acid encoding SEQ ID NO: 139 The performance of the plastid/performance card was co-transfected.

The bispecific antibody was purified by protein A chromatography using supernatants of HEK293 suspension cells encoding L- and H-chains that express transient transfections of plastids (see above). Subsequently, size exclusion chromatography (SEC) was applied to obtain bispecific antibodies free of aggregates or contaminants. An example of the purity and composition of the purified blood brain barrier-swirl module is shown in Figure 8 as the SEC profile and SDS PAGE.

Example 15 Bispecific hapten binds to the blood-brain barrier-snap module simultaneously binds to haptenated payload and blood-brain barrier receptor

In order to achieve the blood-brain barrier-shuttle function of bispecific antibodies, it must bind to the blood-brain barrier receptor on the endothelial cells of the blood-brain barrier and the haptenation to be shuttled. load. To assess this functionality of the hapten-binding bispecific antibody as reported herein, simultaneous cell surface and payload binding was resolved by FACS analysis. For these analyses, cell binding of the blood-brain barrier-shuttle module (=bispecific antibody) was detected by phycoerythrin-labeled IgG-recognizing secondary antibodies. Simultaneous payload binding was detected by applying a haptenated fluorescent payload (digocinyl Cy5; DIG-Cy5). The results of FACS analysis using hCMEC/D3 cells as TfR-expressing BBB-derived cell lines and Dig-Cy5 as fluorescent payloads are shown in Figure 9: two transferrin receptors as indicated by anti-IgG-PE related signals The binding bispecific antibody binds to hCMEC/D3. Similarly, as indicated by the signal evoked by cell-associated Cy5, the two bispecific antibodies also bind to and simultaneously bind to Dig-Cy5. Comparison of the signal intensity between the (high affinity) TfR1 bispecific antibody and the (lower affinity) TfR2 bispecific antibody indicates (as expected) on cells with high affinity compared to the medium affinity bispecific antibody Higher signal strength. A control bispecific antibody that recognizes an antigen that is not present in detectable amounts on hCMEC/D3 (CD33-Dig) does not (as expected) produce an anti-IgG antibody or a signal related to Dig-Cy5.

These results show that the bispecific hapten binds to the blood brain barrier-scission module to specifically bind to its target on the surface of endothelial cells. In addition, these bispecific antibodies simultaneously bind to the haptenation payload and thereby direct it to endothelial cells of the blood brain barrier.

Example 16 Receptor binding mode of the blood-brain barrier-shuttle module affecting the release of endothelial cells from the brain

I used brain endothelial cells (hCMEC/D3) to study cell binding and transcytosis of the shuttle module as reported herein. The aforementioned studies (Crepin et al, 2010; Lesley et al, 1989, WO 2012/075037, WO 2014/033074) report that the valency and affinity of TfR-binding antibodies affect endothelial cells bound to the blood-brain barrier, and the endothelium via the blood-brain barrier. Cell transcytosis and the release of endothelial cells from the blood-brain barrier. To study cell binding and transcytosis in hCMEC/D3, hCMEC/D3 cells cultured on filter inserts were conjugated to bispecific or parental antibodies (no hapten-conjugated scFv) The plants were incubated at 37 ° C for 1 hour at the top. The cell monolayers were washed three times at room temperature (400 μl) and bottom lateral (1600 μl) in serum-free medium for 3-5 minutes each. The total wash volume was collected to monitor the efficiency of removal of unbound ligand or antibody. Pre-warmed medium was added to the top chamber and the filter was transferred to fresh 12-well plates (blocked overnight with PBS containing 1% BSA) containing 1600 μl pre-warmed medium. At this time, the cells were dissolved in 500 μl of RIPA buffer (Sigma, Munich, Germany, #R0278) to determine specific uptake. The remaining filters were incubated at 37 ° C and samples were collected at various time points to determine top and/or bottom lateral release. The antibody content in the samples was quantified using a highly sensitive IgG ELISA. The results of these analyses are shown in Figure 10: The high affinity bivalent anti-TfR antibody (TfRl) becomes efficaciously bound to the cells but not released to the apical or basal compartment. In the same manner, a bispecific antibody containing a high affinity TfR binding site (TfR1-Dig, TfR1-Bio) becomes effective to bind to cells but not to the top or bottom outer compartment. In contrast, a bivalent anti-TfR antibody (TfR2) with reduced affinity becomes effective for binding to cells and is then released over time to the top or bottom outer compartment. A bispecific antibody containing a reduced affinity bivalent TfR binding site (TfR2-Dig, TfR2-Bio) also becomes effective to bind to the cells and is released to the apical or basal compartment to the same extent as the parent antibody. A control bispecific antibody (CD33-Dig, CD33-Bio) that binds to an antigen not present on hCMEC/D3 does not bind to such cells and therefore does not release to the top or bottom outer compartment over time.

Example 17 A blood-brain barrier with a reduced affinity for the TfR shuttle - a shuttle module spans endothelial cells and supports transcytosis and releases haptenated payload

Cellular endothelial cells (hCMEC/D3) were used to study cell binding and transcytosis of the haptening payload, which forms a non-covalent complex with the hapten in combination with the blood-brain barrier-shuttle module. To assess whether payload transcytosis can be achieved by a hapten-associated blood-brain barrier-shuttle module (bispecific antibody) as reported herein for non-covalent complex payloads, hCMEC/ in trans-well systems D3 cells are exposed as by this article The haptenated payload of the reported bispecific antibody complex (see previous examples for the exemplary construct) was allowed to allow for TfR binding for one hour. After removal of the shuttle and payload by washing (see Example 15), the binding molecules were monitored over time (0 to 5 hours after the start of the experiment = washing step) in a similar manner as described for the shuttle module in Example 15. Internalization, intracellular sorting, transcytosis, and payload release. The payload used in the current example is a single haptenized DNA that becomes non-covalently complexed at a 2: 1 (mole) ratio when incubated with a bispecific antibody as reported herein, as shown in Figure 11A. The presence of a net load in the cell extract, the top and bottom outer compartments can be detected and quantified by qPCR. For example, as a net load, the end of the single biotinylated or single digoxin double strand DNA 50 mer (SEQ ID NO: 140) and the two PCR primers PrFor (SEQ ID) used for the net load quantification on the Roche LightCycler NO: 141) and PrRev (SEQ ID NO: 142) are shown in Figure 11A. The results of these analyses (Fig. 11B) indicate that the non-covalently linked haptenated payload binds to the cells, internalizes and is subsequently released into the top and bottom outer compartments. Binding and subsequent release are mediated by TfR in conjunction with the blood-brain barrier-shuttle module, and if CD33 is bound to a control bispecific antibody, neither binding to cells nor detection of release is detected. In addition, neither the binding to the cells nor the detection of release was detected when the haptenized payload of the non-bispecific antibody was applied. Transcytosis of a net load of non-covalent complexes observed by a bispecific antibody and a corresponding hapten-loaded digoxin binding site and a biotin binding site. This shows that different haptens can be used to design a non-covalent bispecific antibody blood-brain barrier-swirl module. Thus, a net load transcytosis across the blood-brain barrier can be achieved using a hapten-binding bispecific antibody directed against a non-covalent complexed haptenated payload.

Example 18 The blood-brain barrier-shuttle module with a high affinity binding site for TfR binds to endothelial cells but does not release from endothelial cells, but still supports transcytosis and release of haptenated payload

Brain endothelial cells (hCMEC/D3) are used to study cell binding for haptenation payload And transcytosis, these payloads can form a non-covalent complex with the hapten in combination with the blood brain barrier-shuttle module in the same manner as previously described in Example 17. HCMEC/D3 cells in the trans-well system were exposed to the blood-brain barrier-snap module (bispecific antibody) complex haptening payload for 1 hour to allow TfR binding, internalization and intracellular sorting, and transformation Endocytosis. The net load is a single haptenated DNA that is non-covalently complexed at a 2: 1 (mole) ratio when incubated with the bispecific antibody, as shown in Figure 11A. Quantification of single biotinylated or monohomolytic double stranded DNA 50 mer payload (SEQ ID NO: 140) by qPCR in cell extract, top and bottom lateral compartments as described in previous Example 17. .

The results of these analyses (Figure 12) indicate that the haptenated payload of the non-covalent complex binds to the cells, internalizes and is subsequently released into the top and bottom outer compartments. This is an unexpected performance because the bivalent high affinity shuttle module itself is not released from the cells. Binding and subsequent payload release are mediated by TfR binding to the bispecific antibody blood-brain barrier-shuttle module, as if CD33 is bound to a control bispecific antibody, neither binding to cells nor detection is detected. To release. In addition, in the case of the hapten-loaded payload of the blood-brain barrier-shuttle module coated with the bispecific antibody, neither binding to the cells nor detection of release was detected. Transcytosis and release of a net load of non-covalent complexes were observed for the digoxin binding site containing the bispecific antibody and the corresponding haptenated payload and the biotin binding site. This indicates that different haptens can be used to design a non-covalent complex of the haptenated payload with the bispecific antibody blood brain barrier-shuttle module. Transdermal transcytosis across cells containing the blood-brain barrier can be achieved by a non-covalent complexed haptening payload of the blood-brain barrier-swirl module (bispecific antibody). Surprisingly, transcytosis does not depend on the release of the shuttle medium itself, since the payload is released even when the shuttle module is not released.

Example 19 The haptening net load in the vesicle compartment is separated from the blood-brain barrier-swirl module

Transcytosis analysis using a high-affinity TfR binding site containing a blood-brain barrier-swirl module (TfR1) that binds to endothelial cells but does not itself release from such cells Unexpected result: The haptenated payload is shuttled across the cell and released into the apical and basal compartments, although the shuttle module itself is still attached to/contained in the cells. Cell binding, uptake and distribution mediated by bispecific antibodies at a net load by confocal microscopy. Therefore, brain endothelial cells (hCMEC/D3) were exposed to a bispecific antibody complex haptenated fluorescent payload (hapten-Cy5 or hapten-DNA-Cy5) and analyzed by confocal fluorescence microscopy. Therefore, hCMEC/D3 cells were seeded onto microscopic glass coverslips and incubated with the 50 nM bispecific antibody complexed haptenated fluorescent payload at 37 ° C for three hours in cell culture medium. The cells were then washed, fixed (4% paraformaldehyde) and the IgG portion of the shuttle module was detected by contrast staining with a secondary antibody that was subsequently bound to ALEXA Fluor 488 with an anti-kappa light chain specific antibody. Images were acquired on a LEICA SP5x confocal microscope using a 100x/1.46NA objective lens with appropriate bandpass filter settings for ALEXAFluor 488 (IgG) and CY5 (hapten-DNA-CY5 payload). The results of these analyses are shown in Figure 13. The complex of the high affinity bispecific antibody and the fluorescently labeled haptenated payload (DNA-Cy5) binds to TfR and is initially localized on the cell surface. Subsequently, it is internalized with its cognate receptor and is present in the cells in the vesicle compartment, ie endosomes and lysosomes. Shortly after internalization (three hours), we observed that the fluorescent signal caused by the shuttle module was substantially separated from the fluorescent signal caused by the haptenization payload. Thus, the non-covalent complex and haptenated payload of the blood-brain barrier-shuttle module as reported herein can be dissolved into different vesicle compartments within the cell. Thus, the payload is released from the shuttle module and can exit the endothelial cells via transcytosis, even though the shuttle module is still bound to the cells/retained in the cells. Intracellular separation of the haptenated payload of the non-covalent complex was observed by digoxin binding and biotin in combination with the blood-brain barrier-swirl module (bispecific antibody) and the corresponding haptenation payload. Thus, different hapten designs can be used to achieve a haptenated payload of payload transcytosis and a non-covalent complex of the blood brain barrier-shuttle module.

Example 20 HeliCar motif amino acid sequence containing peptide YY

Peptide YY is a short (36 amino acid) peptide that responds to food release in cells in the ileum and colon. In humans, it seems to have a loss of appetite. The most common form of the circulating PYY is PYY _3-36 , which binds to the Y2 receptor (Y2R) of the receptor Y family. PYY is present in L cells in the mucosa of the gastrointestinal tract, especially the ileum and colon. In addition, a small amount of PYY (about 1 to 10%) is present in the esophagus, stomach, duodenum, and jejunum. During the cycle, the PYY concentration increased after food intake and decreased during fasting. PYY exerts its effects by the NPY receptor; it inhibits gastric movement and increases water and electrolyte absorption in the colon. PYY and PYY mimics have been used to address obesity.

PYY is modified to include the HeliCar motif amino acid sequence and is complexed with an anti-HeliCar motif amino acid sequence antibody to obtain the advantages of the pharmacokinetic properties of the antibody and to avoid the inherent instability of PYY.

Non-covalent complex formation

Structural studies of PYY _3-36 peptides (Nygaard, R. et al., Biochem. 45 (2006) 8350-8357; SEQ ID NO: 143) reveal the helical motif of the central amino acid (HeliCar-like motif amino acid sequence) ). Since the N-terminal isoleucine and the modified C-terminus have been described as essential for the functional activity of the peptide, the central helix is modified to reflect the amino acid in the HeliCar motif amino acid sequence.

PYY(3-36)3 36(SEQ ID NO.143)IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRYNH2 HeliCar Primitive A HL E N EVA RL KK PYY_HeliCar IKPEAPGEDASPE AHLANEVARL HYLNLVTRQRYNH2 (SEQ ID NO: 144) (YNH2 = tyrosine guanamine)

A fully IgG1 anti-HeliCar motif amino acid sequence antibody was produced in HEK293 cells by transfecting two plastids containing the heavy and light chain variable regions of the inserted vector, which contain constant human IgG1, respectively. And a constant human lambda domain. The anti-HeliCar motif amino acid sequence antibody (0019) was purified by standard procedures using Protein A chromatography. Mass spectrometry experiments confirmed the identity of antibody 0019.

A complex of antibody 0019 and modified PYY peptide PYY_HeliCar was obtained in vitro by applying a small excess of peptide to the antibody solution. Complex 0052 is formed. The stoichiometric amount of the complex was determined by SEC-MALLS analytical assay to complex 1.6 peptides on a bivalent antibody.

The effect of antibody 0019, PYY(3-36) wild type, PYY_HeliCar, and complex 0052 on the Y2Receptor family was tested.

As demonstrated (Hoffmann, E. et al., J. Cont. Rel. 171 (2013) 48-56.), antibody-complexed peptides have an extended in vivo half-life. In addition and unexpectedly, the complex demonstrates a slightly better affinity of the NPY2R receptor than the non-complex peptide; the antibody stabilizes the polypeptide and A peptide is provided in a fixed biologically active configuration.

Covalent complex formation (covalent disulfide bond)

In order to enhance the in vitro and in vivo stability of a complex of an anti-HeliCar amino acid sequence antibody antibody and a compound containing a HeliCar motif amino acid sequence, a method of forming a disulfide bridge upon binding has been used.

The first step is a specific recognition step (high affinity interaction), i.e., formation of a compound containing an HeliCar motif amino acid sequence - an anti-HeliCar motif amino acid sequence antibody complex. Thereafter, in the second step, the disulfide bridge is spontaneously reorganized to form a covalent complex with improved stability.

Since the 12-mer peptide (HeliCar motif amino acid sequence) is a relatively rigid entity (at least when complexed by a specific anti-HeliCar motif amino acid sequence antibody), it has been found that a structurally specific disulfide bridge must be used. design. Since complex formation and subsequent covalent coupling are between two recombinantly produced entities, it is not necessary to have free cysteine residues for the artificial cysteine residues introduced to form a covalent disulfide bond. The form is produced, but is expressed in a reduced amount, i.e., bound to free cysteine or homocysteine amino acid.

It is important to introduce the position of the artificial free cysteine residue in the amino acid sequence of the variable domain of the anti-HeliCar motif amino acid sequence antibody. The non-exposed cysteine in the antibody variable domain amino acid sequence has a greater probability of exhibiting free cysteine (no binding), while the exposed cysteine residual gene close to the binding pocket is associated with an additional moiety (eg The cysteine binding steric hindrance of free cysteine) abolishes the binding of the 12-mer peptide (HeliCar motif amino acid sequence).

a) complex with a fluorescent compound containing a HeliCar motif amino acid sequence

In order to identify suitable locations with the lowest steric risk and reduced affinity, different positions have been tested for the introduction of artificial cysteine residues in the HeliCar motif amino acid sequence. The cysteine residue has been introduced at the C-terminus of the 12mer (HeliCar motif amino acid sequence) to keep the major portion of the paratope unchanged. The peptide has been synthesized and fused to a fluorescent motif.

Wild type: AHLENEVARLKK (SEQ ID NO: 145)

Cysteine variant 1: AHLENEVARCKK (SEQ ID NO: 146) -> AHLENEVARCKK (5-Fluo)-OH

Cysteine variant 2: AHLENEVARLCK (SEQ ID NO: 147) -> AHLENEVARLCK (5-Fluo) - OH x TFA

On the antibody, a structural design has been made to allow the formation of disulfide bridges for the two design peptides, each of which includes cysteine in a different 3D environment.

The 12-mer helical peptide AHLENEVARLKK (SEQ ID NO: 145, HeliCar motif amino acid sequence) was mimicked into the VH and VH domains. At the C-terminus of the peptide, residues L10 and K11 were identified as possible positions, and in the light chain variable domain, positions N55 and G51 according to the light chain number of Kabat were identified.

The heavy chain variable domain of the anti-HeliCar motif amino acid sequence antibody (0019) has an amino acid sequence: (SEQ ID NO: 148).

The light chain variable domain of the anti-HeliCar motif amino acid sequence antibody (0019) has an amino acid sequence: (SEQ ID NO: 149).

The light chain variable domain N55C variant of the anti-HeliCar motif amino acid sequence antibody (0155) has an amino acid sequence: (SEQ ID NO: 150).

The light chain variable domain N51C variant of the anti-HeliCar motif amino acid sequence antibody (0157) has an amino acid sequence: (SEQ ID NO: 151).

i) covalent conjugate of a compound containing a HeliCar motif amino acid sequence with antibody 0155

Bivalent antibody 0155 is expressed in HEK293 cells similar to the parent molecule Y2R(bck)-0019 without free cysteine. The modified antibody was purified using the same protocol as used for antibody 0019. Mass spectrometry showed that the mass of the deglycosylated antibody determined experimentally was 142,001 Da. This is more than 259Da than the calculated quality. The reduced chain has an experimentally determined mass of 48,167 Da (complete heavy chain, calculated value 48, 168 Da, Cys = SH, C-Term = -K) and 22,720 Da (complete light chain, N55C, calculated 22, 720 Da, Cys = SH). The sequences of the chains are confirmed after reduction.

Antibody 0155 was coupled to the HeliCar motif amino acid sequence cysteine variant 2 using a 2.5 molar excess of the compound containing the HeliCar motif amino acid sequence in 100% DMF to form covalent complex 0156.

On the SDS page (denaturing conditions, see Figure 14), only fluorescence was found on antibody 0155; under reducing conditions, only small peptides were visible.

result:

The covalent attachment of a fluorescent compound containing a HeliCar motif amino acid sequence to an anti-HeliCar motif amino acid sequence antibody was successful. A total of approximately 43% of the anti-HeliCar motif amino acid sequence antibody was covalently bound to two HeliCar motif amino acid sequences, and approximately 40% of the anti-HeliCar motif amino acid sequence antibody was covalently bound to a HeliCar motif amino acid. Sequence, and about 17% anti-HeliCar motif amino acid sequence was not bound.

The conjugate modification comprising two HeliCar motif amino acid sequences is up to about 50%. This substance has not been considered for quantification. Antibodies that do not have a HeliCar motif amino acid sequence contain two modifications of about 128 Da as determined for the starting material. Binding to a HeliCar nucleoside amine The antibody to the acid sequence has only one modification of about 128 Da.

Ii) a covalent conjugate of a compound containing a HeliCar motif amino acid sequence with antibody 0157

Similar to antibody 0155 is antibody 0157, which is typically expressed as a cysteylated form. Mass spectrometry showed that the mass of the deglycosylated antibody determined experimentally was 141,863 Da. This is more than the calculated quality of 3Da. The antibody is present primarily in the form of a mono-cysteine deuterated form or a di-hypercysteine deuterated form. The reduced chain has an experimentally determined mass of 48,168 Da (complete heavy chain, calculated value 48, 168 Da, Cys = SH, C-Term = -K) and 22,777 Da (complete light chain, N51C, calculated 22, 777 Da, Cys = SH). The sequences of the chains are confirmed after reduction.

Coupling of antibody 0157 with the HeliCar motif amino acid sequence cysteine variant 1 does not produce the desired covalent complex. Fluorescence was not found in the lanes, but was shown in the reference lanes that should be negative in this experiment (see Figure 15).

Antibody 0157 was incubated with the HeliCar motif amino acid sequence cysteine variant 1. Control antibody 0019 was incubated with the same HeliCar motif amino acid sequence cysteine variant 1.

result:

The covalent attachment of a fluorescent compound containing a HeliCar motif amino acid sequence to an anti-HeliCar motif amino acid sequence antibody was unsuccessful. Without being bound by this theory, it is assumed that in this case the antibody in the binding pocket is caspase too deep to allow efficient binding of the fluorescent compound containing the HeliCar motif amino acid sequence and transfer of the nucleophilic thiol group at the appropriate position to attack C51.

b) complexed with a recombinant polypeptide containing a HeliCar motif amino acid sequence

The HeliCar-based approach is particularly attractive when considering the formation of covalent complexes with recombinantly produced polypeptides containing the HeliCar motif amino acid sequence.

The binding of antibody 0155 containing the VL-N55C mutation to the HeliCar motif amino acid sequence cysteine variant 1 (AHLENEVARLCK; SEQ ID NO: 146) compared to the substitution (having the HeliCar motif amino acid sequence cysteamine) Acid variant 2 (AHLENEVARCKK; The G51C) on VL of SEQ ID NO: 147) was much better, and the binding of 0155 to the polypeptide containing the HeliCar motif amino acid sequence cysteine variant 1 was further investigated. A polypeptide containing the HeliCar motif amino acid sequence cysteine variant 1 (AHLENEVARLCK; SEQ ID NO: 146) was fused to the N-terminus.

Pseudomonas exotoxin containing the HeliCar motif amino acid sequence cysteine variant 1 (0236; SEQ ID NO: 152) having a C-terminal amino acid residue deletion has been produced in E. coli. The molecule LR8M was purified using anion exchange chromatography and a combination of SEC (see for example WO 2011/032022).

Antibody 0155 is covalently bound to a Pseudomonas aeruginosa exotoxin molecule LR8M containing the HeliCar motif amino acid sequence cysteine variant 1 having a C-terminal amino acid residue deletion of SEQ ID NO:152. The SEC chromatogram is shown in Figure 16. The binding efficiency of the unreduced samples was analyzed by SDS-CE, Cariper (see Figure 17).

A total of about 4% of the anti-HeliCar motif amino acid sequence antibody is covalently bound to two SEQ ID NO: 152 polypeptides, and about 41% of the anti-HeliCar motif amino acid sequence antibody is covalently bound to a SEQ ID NO: 152 polypeptide, And about 55% of the anti-HeliCar motif amino acid sequence was not bound.

In summary, anti-HeliCar motif amino acid sequence monoclonal antibodies can be used to recognize complex peptides, small molecules with peptide linking groups, and recombinant proteins with high affinity of the 12-merHeliCar motif amino acid sequence. A peptide having a tendency to fold into a helix can be modified to mimic the original 12-mer HeliCar motif amino acid sequence AHLENEVARLKK (SEQ ID NO: 145) and thereafter can be complexed with an anti-HeliCar motif amino acid sequence monoclonal antibody. In addition to high affinity complexation, a stable disulfide bond can be formed with a cysteine variant containing cysteine SEQ ID NO: 145 and a modified anti-HeliCar motif amine group containing cysteine in the CDR. Acid sequence antibodies are covalently bound. Recombinant proteins expressed by different systems can then be combined in vitro without the use of specific reaction conditions but by spontaneous disulfide bridge shuffling.

Example 21 Binding to a bispecific antibody from BrdU complexed with a payload containing BrdU

SEC-MALLS analysis was used to assess whether transferrin receptor (TfR)- and bromodeoxyuridine (BRDU)-binding bispecific antibodies (bsAb) are capable of binding to the net load containing BRDU and to what extent. Therefore, BRDU-DNA was added to the TfR-BRDU bsAb at a stoichiometric ratio of 2:1 (350 μg; 2.5 mg/ml) and incubated for 30 minutes at room temperature to form a bsAb/net load complex. We prepared a free bispecific antibody (2.5 mg/ml) and free BRDU-DNA (3.2 mg/m) as a control reagent. BRDU-DNA (BRDU-ACC AAG CCT AGA GAG GAG CAA TAC AAC AGT ACA TAT CGC GTG GTA AGC GT; SEQ ID NO: 153) contains one BRDU per DNA molecule at the 5' end of the DNA. The complex and control reagents were stored at -80 °C until analysis.

The resulting complex and control reagents were subjected to SEC-MALLS analysis to identify and characterize free bispecific antibodies, free payload, and complexes of the two. SEC-MALLS analysis was performed on a Dionex Ultimate 3000 HPLC equipped with Wyatt miniDawnTREOS/QELS and Optilab rEX detectors. The analyte was dissolved in PBS buffer pH 7.4 at 1 mg/ml, applied to a Superdex 200 10/300 GL column at a flow rate of 0.5 ml/min and lysed with PBS buffer pH 7.4 for 60 minutes.

The results of these analyses (shown in Figure 18) indicate that the DNA containing BRDU forms a defined complex with the bispecific antibody. These composites with a MW of 244.9 kDa were eluted from the column (Fig. 18A) and exhibited a hydrodynamic radius of 6.8 nm (Fig. 18B), and the stoichiometric ratio was calculated to be approximately two (1.8) per bispecific antibody molecule. DNA molecule. In contrast, a free bispecific antibody with a MW of 215.4 kDa was detected and its hydrodynamic radius was measured at 6.2 nm. The MW of free BRDU-DNA was detected to be 16.4 kDa.

Thus, a complex containing BRDU-rich DNA and stoichiometrically bound by an anti-TfR/BRDU bispecific antibody to produce a 2:1 molar ratio is shown.

Example 22 Biotin-binding bispecific antibody binds to biotin-containing IgG

To analyze whether TfR/biotin bispecific antibodies bind to single biotinylated To what extent is full-length IgG and binding, an anti-TfR/biotin bispecific antibody is added to an anti-TfR/biotin bispecific antibody with an IgG homotyped single biotinylated antibody that specifically binds to pTau at a 2:1 stoichiometric ratio (300 μg, 1.3 mg/ml). The primed anti-pTau antibody, BIO-pTau), and the mixture was incubated for 30 minutes at room temperature (formation of a bispecific antibody-net load complex). Single biotinylated IgG was produced by making an Avi-tagged IgG derivative at the C-terminus of one of the IgG-type 杵-doped dimeric antibodies. The Avi tag binds to a biotinase in a defined manner.

As a specific control for complex formation, an anti-TfR/digoxigenin bispecific antibody was mixed with BIO-pTau. As another control reagent, an aliquot of both the free bispecific antibody and the free BIO-pTau was prepared. The complex and control reagents were stored at -80 °C until analysis.

The resulting complex was subjected to SEC-MALLS analysis to identify and characterize free bispecific antibodies, free BIO-pTau, and complexes thereof. SEC-MALLS analysis was performed on a Dionex Ultimate 3000 HPLC equipped with Wyatt miniDawnTREOS/QELS and Optilab rEX detectors. The analyte was dissolved in PBS buffer pH 7.4 at 1-2 mg/ml, applied to a Superose 6 10/300 GL column at a flow rate of 0.5 ml/min and lysed with PBS buffer pH 7.4 for 60 minutes.

The results of these analyses (shown in Figure 19) indicate that BIO-pTau forms a defined complex with the bispecific antibody. These composites having a MW of 501 kDa were dissolved from the column (Fig. 19B) and exhibited a hydrodynamic radius of 8.0 nm (Fig. 19A). In contrast, a free bispecific antibody with a MW of 205 kDa was detected and its hydrodynamic radius was measured at 6.2 nm. The free BIO-pTau with a MW of 150 kDa was detected and its hydrodynamic radius was measured at 5.5 nm.

The complex is specifically formed by the interaction between biotin and the biotin-binding portion of the bispecific antibody because the digoxin-binding bispecific antibody does not form a complex with BIO-pTau.

Example 23 Transfer of a complex of biotinylated anti-pTau antibody and anti-TfR/biotin bispecific antibody Endocytosis

To analyze whether anti-TfR/biotin bispecific antibodies contribute to the transcytosis of the full-length antibody payload and to what extent, anti-TfR/biotin bispecific antibodies (anti-TfR) were formed as described in Example 22. /Biotin bsAb-1 and anti-TfR/biotin bsAb-2) complex with BIO-pTau and transcytosis analysis as described above (eg, Example 18). As a control for non-specific transcytosis, the anti-CD33/biotin bispecific antibody complex with BIO-pTau and free BIO-pTau were tested in parallel. Samples of the top and bottom outer compartments and samples of cell lysates were taken after 0, 1, 2, 3, 4 and 5 hours after loading the cells. The loading is always 3.8 μg/ml.

The amount of biotinylated anti-pTau antibody was measured by ELISA. Therefore, the pTau protein was plated at 500 ng/ml onto a NUNC Maxisorb white 384-well culture dish at 2-8 ° C overnight or at room temperature for one hour. Plates were blocked with PBS containing 2% BSA and 0.05% Tween 20 for at least one hour. A sample diluted up to 1/729 fold in PBS containing 0.5% BSA and 0.05% Tween 20 was applied for 1.5-2 hours, followed by poly-HRP40-Fitzgerald for 30 minutes, and Super Signal ELISA Pico substrate (Thermo Scientific) was carried out for 10 minutes at room temperature. Standard dilutions of BIO-pTau antibody (100 ng/ml - 0.5 pg/ml) were analyzed on the same plate. Between successive incubation steps, the plates were washed with PBS containing 0.1% Tween 20.

The results of these transcytosis assays (Figure 20) show that binding of BIO-pTau to anti-TfR/biotin bsAb-1 or anti-TfR/biotin bsAb-2 mediates efficient endocytosis and subsequently BIO-pTau Transfer to the bottom outer side and back to the top compartment. In contrast, neither BIO-pTau nor anti-CD33/biotin bispecific antibody complex or free BIO-pTau was efficiently endocytosed or transcytosed, indicating that the observed transcytosis was by anti-TfR/biotin Bispecific antibodies are caused by specific binding to TfR on the cell surface.

Example 24 Bispecific anti-human Tau (pS422) / biotin antibody and biotinylated anti-TfR antibody Fab Transcytosis of a complex of fragments

Similar to the examples presented above, transcytosis of a non-covalent complex of a bispecific anti-human Tau (pS422)/biotin antibody with a biotinylated anti-TfR antibody Fab fragment has been elucidated. The results are presented in Figure 21. For analysis, human Tau (pS422) was immobilized on a culture plate, and anti-human CH2 was used as a secondary antibody and detected using an anti-digoxigenin antibody POD conjugate.

<110> Swiss company Herfo Monlaro AG

<120> Humanized anti-Tau (pS422) antibody brain shuttle and its use

<130> P32183

<150> EP14174042

<151> 2014-06-26

<160> 153

<170> PatentIn version 3.5

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<223> rbHCfinal.up

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<213> Mus musculus

<400> 67

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<213> Mus musculus

<400> 68

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<213> Mus musculus

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<400> 86

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<400> 96

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<400> 98

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<400> 99

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<400> 100

<210> 101

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<400> 101

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<211> 7

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<400> 102

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<400> 103

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<400> 109

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<400> 110

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<400> 111

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<400> 112

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<400> 113

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<400> 114

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<400> 115

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<400> 116

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<400> 117

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<213> Mus musculus

<400> 118

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<400> 119

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<400> 120

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<220>

<223> Humanized heavy chain variable domain

<400> 129

<210> 130

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Humanized light chain variable domain

<400> 130

<210> 131

<211> 119

<212> PRT

<213> Mus musculus

<400> 131

<210> 132

<211> 107

<212> PRT

<213> Mus musculus

<400> 132

<210> 133

<211> 5

<212> PRT

<213> Mus musculus

<400> 133

<210> 134

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> LC anti-TfR1 antibody

<400> 134

<210> 135

<211> 705

<212> PRT

<213> Artificial sequence

<220>

<223> HC anti-TfR1 antibody binding to scFv anti-digoxigenin antibody

<400> 135

<210> 136

<211> 706

<212> PRT

<213> Artificial sequence

<220>

<223> HC anti-TfR1 antibody binding to scFv avidin antibody fragment

<400> 136

<210> 137

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> LC anti-TfR2 antibody

<400> 137

<210> 138

<211> 705

<212> PRT

<213> Artificial sequence

<220>

<223> HC anti-TfR2 antibody binding to scFv anti-digoxigen antibody fragment

<400> 138

<210> 139

<211> 725

<212> PRT

<213> Artificial sequence

<220>

<223> HC anti-TfR2 antibody binding to scFv avidin antibody fragment

<400> 139

<210> 140

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> End-single biotinylated or single-disepine double-stranded DNA 50 mer payload

<220>

<221> misc_structure

<222> (1)..(1)

<223> n=Biotin or Digoxin

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g or t

<400> 140

<210> 141

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PrFor

<400> 141

<210> 142

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PrRev

<400> 142

<210> 143

<211> 34

<212> PRT

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> X = tyrosine amide

<400> 143

<210> 144

<211> 34

<212> PRT

<213> Artificial sequence

<220>

<223> PYY3-36 helicar

<220>

<221> MISC_FEATURE

<222> (34)..(34)

<223> X = tyrosine amide

<400> 144

<210> 145

<211> 12

<212> PRT

<213> Saccharomyces Cerevisia

<400> 145

<210> 146

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> helicar primitive variant 1

<400> 146

<210> 147

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> helicar primitive variant 2

<400> 147

<210> 148

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-helicar antibody VH

<400> 148

<210> 149

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-helicar antibody VL

<400> 149

<210> 150

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-helicar Antibody VL55C

<400> 150

<210> 151

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> anti-helicar antibody VL51C

<400> 151

<210> 152

<211> 244

<212> PRT

<213> Artificial sequence

<220>

<223> fused to Pseudomonas exotoxin LR8M and having a GGG-peptide linking group and C-terminal K-deleted helicar motif amino acid sequence cysteine variant 1

<400> 152

<210> 153

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> BRDU-DNA conjugate

<220>

<221> misc_RNA

<222> (1)..(1)

<223> BRDU

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g or t

<400> 153

Claims (28)

Use of a non-covalent complex specifically binding to a haptenated antibody of human Tau (pS422) and an anti-blood brain barrier receptor/hapten bispecific antibody for use in manufacturing to bind the specificity to An antibody to human Tau (pS422) transmits an agent that passes through the blood-brain barrier. Use of a bispecific antibody that specifically binds to human Tau (pS422) and a hapten, and a non-covalent complex of a haptenized anti-blood-brain barrier receptor antibody, which is used in the manufacture to bind the specificity to An antibody to human Tau (pS422) transmits an agent that passes through the blood-brain barrier. A use comprising a non-covalent complex that specifically binds to human Tau (pS422) and a bispecific antibody that specifically binds to a specific binding to human Tau (pS422) a first binding specificity of a hapten of the haptenated antibody and a second binding specificity specific for binding to a blood brain barrier receptor, wherein the haptenized antibody that specifically binds to human Tau (pS422) is This first binding specificity of the bispecific antibody specifically binds to the agent for transporting the antibody that specifically binds to human Tau (pS422) through the blood brain barrier. A use comprising a non-covalent complex of a haptenated antibody and a bispecific antibody that specifically binds to a blood brain barrier receptor, the bispecific antibody having a first binding specificity specifically binding to human Tau (pS422) Sexually and specifically binds to a second binding specificity of the hapten of the haptenated antibody that specifically binds to a blood brain barrier receptor, wherein the haptenized antibody that specifically binds to the blood brain barrier receptor The first binding specificity specifically binds to the bispecific antibody, and the non-covalent complex is used to produce an agent for transporting the antibody that specifically binds to human Tau (pS422) through the blood brain barrier. The use of any one of claims 1 to 4, wherein the haptenated antibody is a biotinylated antibody or a digoxigenylated antibody. The use of any one of claims 1 to 4, wherein the blood brain barrier receptor is transferrin receptor (TfR) or low density lipoprotein receptor associated protein 8 (LRP8). The use of any one of claims 1 to 4, wherein the bispecific antibody comprises a) a binding site for the hapten of the haptenated antibody and a binding site for the blood brain barrier receptor Or b) two binding sites for the hapten of the haptenated antibody and a binding site for the blood brain barrier receptor, or c) a binding to the hapten of the haptenated antibody a site and two binding sites for the blood brain barrier receptor, or d) two binding sites for the hapten of the haptenized antibody and two binding sites for the blood brain barrier receptor Or e) a binding site for the hapten of the haptenated antibody and a binding site for human Tau (pS422), or f) two binding sites for the hapten of the haptenated antibody a binding site for human Tau (pS422), or g) a binding site for the hapten of the haptenized antibody and two binding sites for human Tau (pS422), or h) Binding sites for the hapten of the haptenated antibody and two for human T a binding site for au (pS422), wherein in the case of b), c), f) and g), one of the heavy chains of the bispecific antibody comprises a hole mutation, and each other chain comprises a 杵Knock mutation. The use of any one of claims 1 to 4, wherein the bispecific antibody has two binding specificities for binding to the hapten of the haptenated antibody (two antigens) Hapten-binding specificity) and specific binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipid) Two binding specificities of protein receptor-associated protein 8 binding specificity). The use of any one of claims 1 to 4, wherein the antibody that specifically binds to human Tau (pS422) i) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and/or ii) Full length human Tau (SEQ ID NO: 02) that does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) specifically binds to serine at position 422. And/or iv) an aggregate of human Tau (SEQ ID NO: 02) that specifically binds to a serine at position 422, and/or v) specifically binds to an amino acid sequence SEQ ID NO: 01 and human Tau with amino acid mutation S422A. The use of any one of claims 1 to 4, wherein the antibody that specifically binds to human Tau (pS422) comprises a) an HVR of SEQ ID NOs: 08, 18 and 10 in the heavy chain variable domain, or b) HVR of SEQ ID NOS: 08, 09 and 10 in the heavy chain variable domain. The use of any one of claims 1 to 4, wherein the antibody that specifically binds to human Tau (pS422) additionally comprises a) an HVR of SEQ ID NOS: 13, 14 and 15 in the light chain variable domain, Or b) HVR of SEQ ID NOS: 12, 05 and 15 in the light chain variable domain. The use of any one of claims 1 to 4, wherein the antibody that specifically binds to human Tau (pS422) comprises a) in the heavy chain variable domain, the HVR of SEQ ID NOS: 08, 18 and 10, and in the light chain variable domain, the HVR of SEQ ID NOS: 13, 14 and 15, or b) In the heavy chain variable domain, the HVR of SEQ ID NOS: 08, 09 and 10, and in the light chain variable domain, the HVR of SEQ ID NOS: 12, 05 and 15, or c) are variable in the heavy chain In the domain, the HVRs of SEQ ID NOS: 08, 09 and 10, and the HVR of SEQ ID NOS: 13, 14 and 15 in the light chain variable domain. The use of any one of claims 1 to 4, wherein the antibody that specifically binds to human Tau (pS422) comprises a) a heavy chain variable domain of SEQ ID NO: 20 and a light chain of SEQ ID NO: a variable domain, or b) a heavy chain variable domain of SEQ ID NO: 19 and a light chain variable domain of SEQ ID NO: 16, or c) a heavy chain variable domain of SEQ ID NO: 19 and SEQ ID NO: The light chain variable domain, or d) the heavy chain variable domain of SEQ ID NO: 21 and the light chain variable domain of SEQ ID NO: 17. A non-covalent complex that specifically binds to a haptenated antibody of human Tau (pS422) and an anti-blood brain barrier receptor/hapten bispecific antibody. A non-covalent complex of a bispecific antibody that specifically binds to human Tau (pS422) and a hapten and a haptenized anti-blood brain barrier receptor antibody. A non-covalent complex comprising a haptenated antibody and a bispecific antibody that specifically binds to human Tau (pS422), the bispecific antibody having specific binding to the half that specifically binds to human Tau (pS422) The first binding specificity and specificity of the hapten of the antigenized antibody binds to a second binding specificity of the blood brain barrier receptor, wherein the haptenized antibody that specifically binds to human Tau (pS422) is This first binding specificity of the specific antibody specifically binds. A non-covalent complex comprising a haptenated antibody and a bispecific antibody that specifically binds to a blood brain barrier receptor, the bispecific antibody having a first binding specificity specifically binding to human Tau (pS422) and a second binding specificity of the hapten that specifically binds to the haptenated antibody that specifically binds to a blood brain barrier receptor, wherein the haptenized antibody that specifically binds to the blood brain barrier receptor is This second binding specificity of the bispecific antibody specifically binds. The non-covalent complex of any one of claims 14 to 17, wherein the haptenated antibody is a biotinylated antibody or a digoxigenin antibody. The non-covalent complex of any one of claims 14 to 17, wherein the blood brain barrier receptor is a transferrin receptor (TfR). The non-covalent complex according to any one of claims 14 to 17, wherein the bispecific antibody comprises a) a binding site for the hapten of the haptenated antibody and a receptor for the blood brain barrier a binding site, or b) two binding sites for the hapten of the haptenated antibody and a binding site for the blood brain barrier receptor, or c) a target for the haptenated antibody a binding site for the hapten and two binding sites for the blood brain barrier receptor, or d) two binding sites for the hapten of the haptenized antibody and two for the blood brain barrier receptor a binding site, or e) a binding site for the hapten of the haptenated antibody and a binding site for human Tau (pS422), or f) two of the half for the haptenated antibody a binding site for the antigen and a binding site for human Tau (pS422), or g) a binding site for the hapten of the haptenized antibody and two binding sites for human Tau (pS422), or h) two binding sites for the hapten of the haptenated antibody and two binding sites for human Tau (pS422), wherein in the case of b), c), f) and g) One of the heavy chains of the bispecific antibody comprises a purine mutation, and each of the other strands contains a purine mutation. The non-covalent complex according to any one of claims 14 to 17, wherein the bispecific antibody has two binding specificities for binding to the hapten of the haptenated antibody (two anti-hapten bindings) Specificity and specific binding to (human) transferrin receptor (two anti-(human) transferrin receptor binding specificity) or low-density lipoprotein receptor-related protein 8 (anti-low-density lipoprotein receptor) The two binding specificities of the relevant protein 8 binding specificity). The non-covalent complex according to any one of claims 14 to 17, wherein the antibody i) which specifically binds to human Tau (pS422) specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 03, and / or ii) does not bind to full length human Tau (SEQ ID NO: 01) at 1 μg/mL, and/or iii) fully binds to full length human Tau (SEQ ID) phosphorylated at serine at position 422 NO: 02), and/or iv) aggregates of human Tau (SEQ ID NO: 02) that specifically bind to phosphorylation at serine at position 422, and/or v) specifically bind to an amine Human Tau having the amino acid sequence SEQ ID NO: 01 and having the amino acid mutation S422A. The non-covalent complex according to any one of claims 14 to 17, wherein the antibody that specifically binds to human Tau (pS422) comprises a) in the heavy chain variable domain, SEQ ID NOS: 08, 18 and HVR, or b) HVR of SEQ ID NOS: 08, 09 and 10 in the heavy chain variable domain. The non-covalent complex according to any one of claims 14 to 17, wherein the antibody that specifically binds to human Tau (pS422) additionally comprises a) in the light chain variable domain, SEQ ID NOS: 13, 14 and HVR of 15 or b) HVR of SEQ ID NOS: 12, 05 and 15 in the light chain variable domain. The non-covalent complex according to any one of claims 14 to 17, wherein the antibody that specifically binds to human Tau (pS422) comprises a) in the heavy chain variable domain, SEQ ID NOs: 08, 18 and HVR of 10, and in the light chain variable domain, HVR of SEQ ID NOS: 13, 14 and 15, or b) in the heavy chain variable domain, HVR of SEQ ID NOS: 08, 09 and 10, And in the light chain variable domain, HVR of SEQ ID NOS: 12, 05 and 15, or c) in the heavy chain variable domain, HVR of SEQ ID NOS: 08, 09 and 10, and in the light HVR of SEQ ID NOS: 13, 14 and 15 in the chain variable domain. The non-covalent complex according to any one of claims 14 to 17, wherein the antibody that specifically binds to human Tau (pS422) comprises a) the heavy chain variable domain of SEQ ID NO: 20 and SEQ ID NO: 17. a light chain variable domain, or b) a heavy chain variable domain of SEQ ID NO: 19 and a light chain variable domain of SEQ ID NO: 16, or c) a heavy chain variable domain of SEQ ID NO: 19 and SEQ ID NO: a light chain variable domain of 17 or d) a heavy chain variable domain of SEQ ID NO: 21 and a light chain variable domain of SEQ ID NO: 17. A non-covalent complex according to any one of claims 14 to 17, which is for use as a medicament. A non-covalent complex according to any one of claims 14 to 17 for use in the treatment of Alzheimer's Disease.